Antimicrobial preparation and uses thereof

ABSTRACT

A method of enhancing the growth of an animal is provided. The method includes causing the animal to ingest or absorb an effective amount of a preparation obtainable by a process including, bringing a tyrosinate moiety into contact with an Fe(III)-containing substance, optionally wherein the tyrosinate moiety is tyrosine and it is brought into contact with the Fe(III)-containing substance in the presence of a base, or wherein the tyrosinate moiety is a tyrosine salt and it is brought into contact with an Fe(III)-containing substance. Methods for inhibiting, reducing, or preventing biofilm formation or buildup on a surface; the treatment of, inhibition of growth of, and inhibition of colonization by, bacteria, both in biological and non-biological environments; disinfecting surfaces, potentiating the effects of antibiotics and other anti-microbial agents, and increasing the sensitivity of bacteria and other microorganisms, to anti-microbial agents are also provided.

CROSS REFERENCE TO RELATED APPLICATION

This applications claims priority to WO 2016/025448 (PCT/US2015/044603)filed Aug. 11, 2015, U.S. Ser. No. 62/296,386 filed Feb. 17, 2016, andU.S. Ser. No. 62/334,746 filed May 11, 2016, all of which areincorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention is in the field of preparations comprising Fe(III)complexes, that have a broad range of antimicrobial and otheractivities, and methods of making and using the preparations.

BACKGROUND OF THE INVENTION

A biofilm is an accumulation of microorganisms (bacteria, fungi, and/orprotozoa, with associated bacteriophages and other viruses) embedded ina polysaccharide matrix and adherent to solid biological or non-bioticsurfaces. Biofilms are medically important, accounting for over 80percent of hospital-acquired microbial infections in the body. Examplesinclude infections of the: oral soft tissues, teeth and dental implants;middle ear; gastrointestinal tract; urogenital tract; airway/lungtissue; eye; urinary tract prostheses; peritoneal membrane andperitoneal dialysis catheters, indwelling catheters for hemodialysis andfor chronic administration of chemotherapeutic agents (Hickmancatheters); cardiac implants such as pacemakers, prosthetic heartvalves, ventricular assist devices, and synthetic vascular grafts andstents; prostheses, internal fixation devices, percutaneous sutures; andtracheal and ventilator tubing. The microorganisms tend to be far moreresistant to antimicrobial agents and to be particularly difficult forthe host immune system to render an appropriate response. Severalbacterial pathogens have been shown to associate with, and in somecases, grow in biofilms, including Legionella pneumophila, S. aureus,Listeria monocytogenes, Campylobacter spp., E. coli O157:H7, Salmonellatyphimurium, Pseudomonas, Vibrio cholerae, S. epidermidis, E. faecalis,and Helicobacter pylon.

Biofilms are remarkably difficult to treat with antimicrobials.Antimicrobials may be readily inactivated or fail to penetrate into thebiofilm. In addition, bacteria within biofilms have increased (up to1,000-fold higher) resistance to antimicrobial compounds, even thoughthese same bacteria are sensitive to these agents if grown underplanktonic conditions.

In addition, bacteria embedded within biofilms are resistant to bothimmunological and non-specific defense mechanisms of the body. Contactwith a solid surface triggers the expression of a panel of bacterialenzymes, which catalyze the formation of sticky polysaccharides thatpromote colonization and protection. The structure of biofilms is suchthat immune responses may be directed only at those antigens found onthe outer surface of the biofilm, and antibodies and other serum orsalivary proteins often fail to penetrate into the biofilm. In addition,phagocytes are unable to effectively engulf a bacterium growing within acomplex polysaccharide matrix attached to a solid surface. This causesthe phagocyte to release large amounts of pro-inflammatory enzymes andcytokines, leading to inflammation and destruction of nearby tissues.Conventional therapy is characteristically ineffective against biofilms,as the minimum inhibitory concentration (MIC) of antimicrobial agentshas been shown to be 10 to 1000 fold greater than for planktonicorganisms (Hoiby, et al., Int J Antimicrob Agents, 35(4):322-32 (2010).

It is an object to provide a preparation (and compositions comprisingthe preparation), methods of making thereof, and uses thereof forinhibiting or preventing biofilm formation or promoting biofilmdissolution from surfaces of interest.

SUMMARY OF THE INVENTION

A preparation comprising an Fe(III) complex, and suitable methods formaking the preparation, has been found to provide a broad range ofactivity, particularly against a diverse array of bacteria.Compositions, articles and products comprising the preparation, are alsodescribed.

Compositions comprising the preparation, and methods and uses employingthe preparation and/or compositions, for inhibiting, reducing, orpreventing biofilm formation or buildup on a surface or to removing,dispersing, reducing, or eradicating biofilm on a surface are disclosed.Accordingly, preparations for inhibiting, reducing, or removing biofilmbuildup in a subject and/or on an article or other item are provided.The preparations are effective against biofilms produced by a wide rangeof microbial species including, without limitation, S. epidermidis, E.faecalis, E. coli, S. aureus, Campylobacter spp. H pylori andPseudomonas, alone, or in combination.

In an embodiment, an article or product, including medical deviceshaving on the surface or dispersed therein one or more of thepreparations as described further below. The surface may be a biologicalsurface (such as a surface of a living human, animal or plant surface,or the surface of a dead or harvested animal or plant), or anon-biological surface including for example, plastics, polymers,biomaterials, and metals.

In another embodiment, the invention provides a preparation for thetreatment of, inhibition of growth of, and inhibition of colonizationby, bacteria, both in biological and non-biological environments.

In a further embodiment, the preparations are used for disinfectingsurfaces, both in biological and non-biological environments, andproducts that have been coated with, or treated by, one or more of thepreparations.

In another embodiment, the preparations are used for potentiating theeffects of one or more antibiotics, increasing the sensitivity ofbacteria (including antibiotic-resistant bacteria) to one or moreantibiotics, and to reversing antibiotic resistance in bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar graph showing biofilm formation by Enterococcusfaecalis at time T=0 in the presence of absence of differentconcentrations of Fe-QA. FIG. 1B is a bar graph showing biofilmformation by Enterococcus faecalis at time T=24 h in the presence orabsence of different concentrations of Fe-QA.

FIG. 2A is a bar graph showing biofilm formation by Staphylococcusepidermidis at time T=0 in the presence of absence of differentconcentrations of Fe-QA. FIG. 2B is a bar graph showing biofilmformation by Staphylococcus epidermidis at time T=24 h in the presenceof absence of different concentrations of Fe-QA.

FIG. 3A is a bar graph showing biofilm formation by Staphylococcusaureus at time T=0 in the presence of absence of differentconcentrations of Fe-QA. FIG. 3B is a bar graph showing biofilmformation by Staphylococcus aureus at time T=24 h in the presence ofabsence of different concentrations of Fe-QA.

FIG. 4A is a bar graph showing the coverage rate of PAO1 Pseudomonasaeruginosa on the surface of a glass slide, comparing Pseudomonas mediumonly as a control, PAO1 Pseudomonas+100 μM Fe-QA treatment, and PAO1Pseudomonas with no Fe-QA (X=Fe-QA). The graph shows that 100 μM Fe-QA(“X”) inhibits the formation of biofilm by P. aeruginosa. FIG. 4B is abar graph showing that Fe-QA inhibits founation of biofilm byUropathogenic E. coli (UPEC). The bar graph shows the coverage rate ofUPEC on the surface of a glass slide compared to a UPEC medium onlycontrol, and UPEC growing in the presence of 0.1 μM, 10 μM, and 100 μMconcentrations of Fe-QA.

FIG. 5 shows quantitatively the difference in the attachment of EPECcells to the plastic well surface in the absence and presence of FeQ bymeasurement of the optical absorbance of crystal violet that wasabsorbed by EPEC cells attached to the surface.

FIGS. 6A-C show chemical structures of how FeQ can be conjugated to anagent that contains a reactive functional group suitable forimmobilizing FeQ, for example, on a surface. FIG. 6A shows theconjugation of FeQ to a calix[4] arene frame that contains aphotoreactive functional group. FIG. 6B shows the conjugation of FeQ toa calix [4] arene frame wherein the photo-reactive functional group ispositioned in a different location on the calix [4] arene frame comparedto the structure of FIG. 6B. FIG. 6C shows the conjugation of FeQ to acalix[4] arene frame functionalized with two thiol groups.

FIGS. 7A and B are chemical structures that illustrate how FeQ can beconjugated via a linker to a substance that binds to a surface. In bothstructures, the linker is spaced between functional groups Y′ and X′,attached to FeQ via Y′ and to hydroxyapatite (HA) via X′. The figuresdiffer in the point of attachment to the quinic acid ligand.

FIG. 8 is a graph showing the average body weight (ABW) of chicken after42 days of growth. The graph compares the ABW at 42 days of chickenchallenged with Campylobacter-infected dirty litter at day 20 andtreated from days 0-42 with FeQ or a preparation obtained by the methodof Example 14 (labelled “FeTyr”) to (i) a standard commercial target (of2.979 kg) labeled “Target”, (ii) a negative control (of 3.437 kg)labeled “CNC” where the chicken were not challenged withCampylobacter-infected dirty litter, and (iii) a positive control (of3.186 kg) labeled “CC” where the chicken were challenged withCampylobacter-infected dirty litter. The graph shows that birdschallenged with Campylobacter-infected dirty litter have higher ABW at42 days compared to the positive control (labeled “CC”) when treatedwith (iv) FeQ at 0.22 g/L in drinking water and FeQ at 0.22 g/kg infeed, labeled “FeQ(W+F)” with an ABW of 3.342 kg, (v) FeQ at 0.22 g/L indrinking water, labeled “FeQ(W)” with an ABW of 3.407 kg, (vi) FeQ at0.22 g/kg in feed, labeled “FeQ(F)” with an ABW of 3.464 kg, (vii) FeQat 0.022 g/L in drinking water, labeled “FeQ(W)” with an ABW of 3.304kg, and (viii) FeTyr at 0.02 g/L in drinking water, labeled FeTyr(W)with an ABW of 3.341 kg.

FIG. 9 is a graph showing the mortality adjusted feed conversion ratio(MFCR) of chicken after 42 days of growth. The graph compares the MFCRat 42 days of chicken challenged with Campylobacter-infected dirtylitter at day 20 and treated from days 0-42 with FeQ or a preparationobtained by the method of Example 14 (labelled “FeTyr”)to (i) a standardcommercial target (of 1.703) labeled “Target”, (ii) a negative control(of 1.563) labeled “CNC” where the chicken were not challenged withCampylobacter, and (iii) a positive control (of 1.679) labeled “CC”where the chicken were challenged with Campylobacter-infected dirtylitter. The graph shows that birds challenged withCampylobacter-infected dirty litter have lower MFCR at 42 days comparedto the positive control (labeled “CC”) when treated with (iv) FeQ at0.22 g/L in drinking water and FeQ at 0.22 g/kg in feed, labeled“FeQ(W+F)” with a MFCR of 1.595, (v) FeQ at 0.22 g/L in drinking water,labeled “FeQ(W)” with a MFCR of 1.560, (vi) FeQ at 0.22 g/kg in feed,labeled “FeQ(F)” with a MFCR of 1.563, (vii) FeQ at 0.022 g/L indrinking water, labeled “FeQ(W)” with a MFCR of 1.612, and (viii) FeTyrat 0.02 g/L in drinking water, labeled FeTyr(W) with a MFCR of 1.577.

FIG. 10 is a graph showing the number of Campylobacter colony formingunits per gram (cfu/g) of bird droppings at day 42. The graph comparesthe cfu/g at day 42 of chicken that were challenged withCampylobacter-infected dirty litter at day 20 and treated from days 0-42with FeQ or a preparation obtained by the method of Example 14 (labelled“FeTyr”) to (i) a negative control labeled “CNC” (with a cfu/g of28,000) where the chicken were not challenged withCampylobacter-infected dirty litter, and (ii) a positive control labeled“CC” (with a cfu/g of 1,280,000) where chickens were challenged withCampylobacter-infected dirty litter at day 21. The graph shows thatbirds treated with FeQ or FeTyr have lower levels of Campylobacter intheir droppings at day 42 when treated with (iii) FeQ at 0.22 g/L indrinking water and FeQ at 0.22 g/kg in feed, labeled “FeQ(W+F)” with acfu/g of 4,860, (iv) FeQ at 0.22 g/kg in feed, labeled “FeQ(F)” with acfu/g of 12,800, (v) FeQ at 0.022 g/L in drinking water, labeled“FeQ(W)” with a cfu/g of 900,000, and (vi) FeTyr at 0.02 g/L in drinkingwater, labeled FeTyr(W) with a cfu/g of 16,600.

FIG. 11 is a graph showing the average number of Campylobacter colonyforming units per gram (cfu/g) of caeca samples at day 42. The graphcompares the cfu/g at day 42 of chicken that were challenged withCampylobacter-infected dirty litter at day 20 and treated from days 0-42with FeQ or a preparation obtained by the method of Example 14 (labelled“FeTyr”) to (i) a negative control labeled “Treatment-1” where thechicken were not challenged with Campylobacter-infected dirty litter,and (ii) a positive control labeled “Treatment-2” where chickens werechallenged with Campylobacter-infected dirty litter at day 21. The graphshows that birds treated with FeQ or FeTyr have lower levels ofCampylobacter in their caeca at day 42 when treated with (iii) FeQ at0.22 g/L in drinking water and FeQ at 0.22 g/kg in feed, labeled“Treatment-3”, (iv) FeQ at 0.22 g/L in water, labeled “Treatment-5”, (v)FeQ at 0.22 g/kg in feed, labeled “Treatment-6”, (vi) FeQ at 0.022 g/Lin drinking water, labeled “Treatment-7”, and (vii) FeTyr at 0.02 g/L indrinking water, labeled “Treatment-8”.

FIG. 12 is shows quantitatively the difference in the attachment of EPECcells to the plastic well surface in the absence and presence of FeDOPA(also referred to as Fe-DOPA) by measurement of the optical absorbanceof crystal violet that was absorbed by EPEC cells attached to thesurface.

FIG. 13 shows 3 bar graphs at 24, 48 and 72 hours of the opticalabsorbance of crystal violet that was absorbed by the EPEC cells thatremained attached to the surface of the plastic well after a maturebiofilm formed by EPEC-pgA⁺⁺ was treated with FeTyr) (shown as “FeY” inFIG. 13) at 100 μM, 150 μM and 200 μM compared to an untreated biofilm(labeled “Control”) in a crystal violet assay.

FIGS. 14A and B show the results of Example 30, which investigateeffects upon antibiotic resistance of a laboratory strain of Pseudomonasaeruginosa (PAO1N) and a mixed population of clinical isolates (PAOMixed), when incubated in Luria-Bertani (LB) media alone, or withdifferent concentrations (34 μM, 100 μM, 200 μM and 340 μM) of FeQ orFePhe. FIG. 14A shows the results with PAO1N cultures. FIG. 14B showsthe results with PAO Mixed cultures.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Aerosol” as used herein refers to any preparation of a fine mist ofparticles, which can be in solution or a suspension, whether or not itis produced using a propellant.

“Biofilm” as used herein refers any group of microorganisms in whichcells stick to each other on a surface.

A “cream” is a viscous liquid or semi-solid emulsion of either the“oil-in-water” or “water-in-oil type”.

An “emulsion” is a composition containing a mixture of non-misciblecomponents homogenously blended together.

“Gel” as used herein is a colloid in which the dispersed phase hascombined with the continuous phase to produce a semisolid material, suchas jelly.

“Cleaning formulation”, as used herein, means a composition suitable forapplication to a surface for removing dirt and oils, for disinfecting,or a combination thereof. Cleaning formulations can be antibacterial,antimicrobial, or both. Cleaning formulations are suitable for use onthe human skin, when none of the components of the composition arepresent at concentrations that cause significant signs of irritationwhen applied to human skin As used herein, “significant signs ofirritation” include erythema, redness, and/or swelling at the site ofinjection or at the site of application, necrosis at the site ofapplication, exfoliative dermatitis at the site of application, andsevere pain that prevents daily activity and/or requires medicalattention or hospitalization. Cleaning formulations can be suitable foruse in the human buccal cavity. Cleaning formulations can be suitablefor use with articles that, subsequent to exposure and optionally withresidual levels of cleaning composition present on and/or in thearticle, will then be contacted with the human skin or other part of thehuman body, such as wherein the article (e.g. a denture) will becontacted with the buccal cavity, or will be contacted with the eye(e.g. a contact lens). Cleaning formulations can be suitable for usewith foodstuffs and/or their packaging and may, for example, be suitablefor cleaning meat products and/or carcasses used in the production ofmeat products. Cleaning formulations may be suitable for cleaningequipment used in food production. Cleaning formulations may be suitablefor use in cleaning medical devices, including implantable medicaldevices. Many other types of cleaning formulations may also be providedby the present invention, further examples of which are discussed infurther sections of this application.

“Chronic wound” as used herein refers to a wound that fails to progressthrough an orderly and timely sequence of repair or a wound that doesnot respond to treatment and/or the demands of treatment are beyond thepatient's physical health, tolerance or stamina Many wounds that arefirst considered to be acute wounds ultimately become chronic wounds dueto factors still not well understood. One significant factor is thetransition of planktonic bacteria within the wound to form a biofilm.

“Inhibition” or “inhibiting” of biofilm formation as used herein refersto a decrease of biofilm associated microorganism formation and/orgrowth.

A “lotion” is a low- to medium-viscosity liquid formulation.

“Oil” as used herein refers to a composition containing at least 95% wt.of a lipophilic substance. Examples of lipophilic substances include butare not limited to naturally occurring and synthetic oils, fats, fattyacids, lecithins, triglycerides and combinations thereof.

An “ointment” is a semisolid preparation containing an ointment base andoptionally one or more active agents.

“Parenteral administration”, as used herein, means administration by anymethod other than through the digestive tract or non-invasive topical orregional routes.

“Patient” or “subject” to be treated and/or used in accordance with anyof the aspect as described herein refers to either a human or non-humananimal such as a primate, non-human primate, laboratory animal, farmanimal, livestock, or a domestic pet. Exemplary animals can optionallyinclude chickens, particularly a meat-type chicken such as broilerchicken, or an egg-laying chicken such as a pullet or hen, or a breederchicken. Also optionally included without limitation are other poultry,such as a turkey, geese, quail or ducks, or livestock, such as cattle,sheep, goats or swine, alpaca, banteng, bison, camel, cat, deer, dog,donkey, gayal, guinea pig, horse, llama, mule, rabbit, reindeer, waterbuffalo, yak, although the skilled person will appreciate that otheranimals, including zoo animals, captive animals, game animals, fish(include freshwater and saltwater fish, farmed fish, and ornamentalfish), other marine and aquatic animals, including shellfish such as,but not limited to, oysters, mussels, clams, shrimps, prawns, lobsters,crayfish, crabs, cuttlefish, octopus, and squid, domestic animals suchas cats and dogs, rodents (such as mice, rats, guinnea pigs, hamsters),and horses, are also included, as well as any other domestic, wild andfarmed animal, including mammals, marine animals, amphibians, birds,reptiles, insects and other invertebrates.

“Pharmaceutically acceptable” as used herein refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals (such as one or more of the animal“patients” or “subjects” as discussed above) without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable salt”, as used herein, refers toderivatives of the compounds defined herein, wherein the parent compoundis modified by making acid or base salts thereof.

“Therapeutically effective” or “effective amount” as used herein meansthat the amount of the composition used is of sufficient quantity toameliorate one or more causes or symptoms of a condition, bacterialcolonization, disease or disorder. Such amelioration only requires areduction or alteration, not necessarily elimination. As used herein,the terms “therapeutically effective amount” “therapeutic amount” and“pharmaceutically effective amount” are synonymous. One of skill in theart can readily determine the proper therapeutic amount.

“Treatment”, “treating”, or “alleviating” as used in connection with adisease or infection refers to an intervention performed with theintention of altering or inhibiting the pathology of a disorder.

II. Compositions and Methods of Use Thereof

Preparations containing the iron complexes, methods of use and benefitsthereof, can be summarized as:

Enhancement of animal growth;

Potentiating the effect of antibiotics and other antimicrobial agents,and addressing antibiotic resistance;

Inhibition of formation, and treatment of preformed, biofilms; treatingmicrobial infections reducing microbial colonization; and disinfectingsurfaces.

Preparations containing the iron complexes can be used to enhance animalgrowth. Numerous examples of this effect are provided in the Examples.

It has been discovered that the preparations containing the ironcomplexes are particularly useful in treating or preventing infection byantibiotic-resistant microorganisms. The preparation may be administeredin order to cause microorganisms to lose their resistance toantibiotics.

Inhibition of Formation, and Treatment of Preformed, Biofilms

In one aspect the preparations containing the iron complexes have abroad range of action in treating and dispersing pre-existing biofilms,and inhibiting the development of biofilms, created by a wide range ofbacterial and other microbial sources. This action is effective in adiverse array of environments.

1. Organisms to be Treated, Inhibited, or Killed

Biofilms are usually found on solid substrates submerged in or exposedto an aqueous solution, although they can form as floating mats onliquid surfaces. Biofilms can form on a myriad of surfaces. “Biofilm” asused herein refers any group of microorganisms in which cells stick toeach other on a surface. For example, biofilms can grow in showers veryeasily since they provide a moist and warm environment for the biofilmto thrive. Biofilms can form inside water and sewage pipes and causeclogging and corrosion. Biofilms on floors and counters can makesanitation difficult in food preparation areas. Biofilms can form incooling- or heating-water systems and are known to reduce heat transferin these systems

One method, or use, includes administering an effective amount of theone or more preparations to inhibit biofilm formations, oralternatively, to reduce and/or remove biofilm formation. The one ormore preparations may be administered alone, or in combination with anantimicrobial agent, such as an antibiotic.

In other embodiments, the method includes contacting a surface with aneffective amount of the one or more preparations, to inhibit biofilmbuildup, reduce built up biofilm, and/or remove built up biofilm.“Contacting” includes, but is not limited to, touching, impregnating,compounding, mixing, integrating, coating, spraying, dipping, flushing,irrigating, and wiping. In certain embodiments, it may be desirable toprovide continuous delivery of one or more preparations to the surfaceor system being treated. The compositions can be used to coat,impregnate, flush, or rinse a surface of tubing or a medical device,especially an insertable medical device. Tubing includes, but is notlimited to, disposable, permanent, and indwelling catheters, long termurinary devices, tissue bonding urinary devices, wound drain tubes,ventricular catheters, endotracheal tubes, breathing tubes, feedingtubes, dairy lines, oil and gas pipeline and drinking water lines. Whenan object is tubing (e.g., dental unit waterline, a dairy line, a foodand beverage processing line, etc.), a composition may be poured intothe tubing and both ends of the tubing clamped such that the compositionis retained within the lumen of the tubing. The tubing is then allowedto remain filled with the composition for a period of time sufficient toremove substantially all of the microorganisms from at least one surfaceof the object, generally, for at least about 1 minute to about 48 hours.Alternatively, tubing may be flushed by pouring a composition into thelumen of the tubing for an amount of time sufficient to preventsubstantial growth of all biofilm embedded microorganisms. Such flushingmay be required only once, or may be required at regular intervals overthe lifetime of use of the tubing. Concentrations of active componentsin a composition may vary as desired or necessary to decrease the amountof time the composition is in contact with a medical device.

The methods allow disinfection, inhibition, or prevention of biofilmformation on the surfaces being treated and reduction of transmission ofbiofilm forming microorganisms from the surface to another surface. Thenumber of the bacterial colony forming units (cfu) on the surface beingtreated with the one or more preparations may be reduced by 50%, by 60%,by 70%, by 80%, by 90% or by 100%, or, the buildup of bacterial colonyforming units on the treated surface may be reduced by 50%, by 60%, by70%, by 80%, by 90% or by 100%.

2. Methods of Administration

In one embodiment, the preparations and formulations, derivativesthereof and combinations thereof for use can be administered topicallyto a subject in need thereof in an effective amount to prevent or treata microbial infection, by inhibiting buildup of biofilm or to reduceand/or remove built up biofilm.

Any suitable topical formulation can be used, for example as describedbelow, including emulsions, lotions, creams, ointments, gels, or foams.

The compositions may be used alone or in combination with knownantimicrobial agents, such as those described further below.

Other modes of administration can include:

Parenteral administration, which may include administration to a patientintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intravitreally, intratumorally,intramuscularly, subcutaneously, subconjunctivally, intravesicularly,intrapericardially, intraumbilically, by injection, and by infusion, forexample as further described.C.1 of this application, below. Parenteraladministration can include the use of formulations as described hereinwhich are formulated for controlled release including immediate release,delayed release, extended release, pulsatile release, and combinationsthereof,.

The preparations can be incorporated into injectable/implantable solidor semi-solid implants, such as polymeric implants.

Enteral administration, including administration in the form of suitableoral dosage forms such as tablets, capsules, solutions, suspensions,syrups, and lozenges,. Optionally, enteral administration may includeadministration of controlled release enteral formulations, includingoral dosage forms, such as capsules, tablets, solutions, andsuspensions, which are formulated for controlled release, includingextended and/or delayed release.

The administration of one or more disinfecting formulations or cleaningformulations.

3. Hospital and Other Environments

The methods and uses may be practiced in the hospital and also in othermedical and non-medical environments in order to address, inhibit,treat, ameliorate and/or disrupt biofilms for the treatment and/orprophylaxis of subjects (including humans and animals) in need thereof.

In other embodiments, the one or more preparations containing the ironcomplexes can be incorporated into coatings used to coat medicaldevices, and other articles.

Suitable coating methods are known in the art. Methods for coatingmedical devices are disclosed for example in U.S. Publication Nos.20030054090 and 20120276280 and U.S. Pat. Nos. 5,879,697, 7,247,338 andU.S. Pat. No. 8,028,646.

In a preferred embodiment, the one or more preparations are combinedwith polymers, and coated on medical devices or other articles. Suitablepolymers include, but are not limited, to poly(lactides);poly(glycolides); poly(lactide-co-glycolides); poly(lactic acid);poly(glycolic acid); poly(lactic acid-co-glycolic acids);polycaprolactones; poly(orthoesters); polyanhydrides;poly(phosphazenes); polyhydroxyalkanoates [includingpoly-3-hydroxybutyrate, poly-3-hydroxybutyrate-co-3-hydroxyvalerate(PHBV), poly-4-hydroxybutyrate,poly-3-hydroxybutyrate-co-4-hydroxybutyrate]; synthetically orbiologically prepared polyesters (including polyesters with one or moreof the following monomeric units: glycolic, lactic; trimethylenecarbonate, p-dioxanone, or ε-caprolactone);poly(lactide-co-caprolactones); polyesters; polycarbonates; tyrosinepolycarbonates; polyamides (including synthetic and natural polyamides,polypeptides, and poly(amino acids)); polyesteramides; poly(dioxanones);poly(alkylene alkylates); polyethers (such as polyethylene glycol, PEG,and polyethylene oxide, PEO); polyvinyl pyrrolidones or PVP;polyurethanes; polyetheresters; polyacetals; polycyanoacrylates;poly(oxyethylene)/poly(oxypropylene) copolymers; polyacetals,polyketals; polyphosphates; (phosphorous-containing) polymers;polyphosphoesters; polyalkylene oxalates; polyalkylene succinates;poly(maleic acids); chitin; chitosan; modified chitosan; collagen; silk;biocompatible polysaccharides; biocompatible copolymers (including blockcopolymers or random copolymers); hydrophilic or water soluble polymers,such as polyethylene glycol, (PEG) or polyvinyl pyrrolidone (PVP), withblocks of other biocompatible or biodegradable polymers, for example,poly(lactide), poly(lactide-co-glycolide, or polycaprolcatone orcombinations thereof, polymers and copolymers of ethylene and propylene,including ultra-high molecular weight polyethylene, ultra-high molecularweight polypropylene, nylon, polyesters such as poly(ethyleneterephthalate), poly(tetrafluoroethylene), polyurethanes,poly(ether-urethanes), poly(methylmethacrylate), polyether ether ketone,polyolefins, Dacron, latex, silicones, polymeric cements, andpoly(ethylene oxide).

In another preferred embodiment, the components of one or morepreparations can be first conjugated with other agents that have anaffinity for, or can react with, a surface, and thereby immobilized on asurface. For example, the components of one or more preparations can betethered to a linkage that can be photo-activated to bind to a surface,or activated via another mechanism.

Examples of devices and articles that can be coated using thecompositions include tubing and other surface medical devices, such asurinary catheter, stents, mucous extraction catheter, suction catheter,umbilical cannula, contact lenses, intrauterine devices, intravaginaland intraintestinal devices, endotracheal tubes, bronchoscopes, dentalprostheses and orthodontic devices, dentures, teeth, surgicalinstruments, dental instruments, tubing, dental water lines, dentaldrain tubes, fabrics, paper, indicator strips (e.g., paper indicatorstrips or plastic indicator strips), adhesives (e.g., hydrogeladhesives, hot-melt adhesives, or solvent-based adhesives), bandages,tissue dressings or healing devices and occlusive patches, and any othersurface devices used in the medical field. Devices may includeelectrodes, external prostheses, fixation tapes, compression bandages,and monitors of various types. Medical devices also include any devicethat may be placed at the insertion or implantation site such as theskin near the insertion or implantation site, and which include at leastone surface which is susceptible to colonization by biofilm embeddedmicroorganisms. In one specific embodiment, a composition is integratedinto an adhesive, such as tape, thereby providing an adhesive, which mayprevent growth or proliferation of biofilm embedded microorganisms on atleast one surface of the adhesive. Medical devices include surfaces ofequipment in operating rooms, emergency rooms, hospital rooms, clinics,and bathrooms. In a particularly preferred embodiment the followingdevices may be coated with the one or more preparations, or one or morecomponents thereof: catheters, including central venous catheters,urinary catheters, dialysis catheters, and indwelling catheters (forexample, catheters for hemodialysis and for administration ofchemotherapeutic agents), cardiac implants including mechanical heartvalves, stents, ventricular assist devices, pacemakers, cardiac rhythmmanagement (CRM) devices, cardiac resynchronization therapy devices(CRTs), and implantable cardioverter defibrillators (ICDs), syntheticvascular grafts, arteriovascular shunts, cerebral spinal fluid shunts,cochlear devices, prosthetic joints, orthopedic implants, internalfixation devices, bone cements, percutaneous sutures, surgical mesh andsurgical patches including hernia repair meshes and patches, breastreconstruction meshes and patches, meshes and patches for breast andface lifts, slings, and meshes and patches for pelvic floorreconstruction, tracheal and ventilator tubing, wound dressings,biological implants (including allografts, xenografts and autografts),penile implants, intrauterine devices, endotracheal tubes, and contactlenses.

Other articles that can be coated include articles for use in rearinganimals. Other articles that can be coated include articles for use inthe process of slaughter and/or processing the carcasses or partsthereof of animals. Other articles that can be coated include articlesfor the preparation and/or containment of food stuffs, includingfoodstuffs comprising raw or cooked meats, eggs, dairy products or otherfood products. The food products may be human and/or animal foodproducts. Additional articles that can be coated include articles forthe preparation and/or containment of drinks.

In another embodiment there is provided a method of disinfecting asurface, or protecting a surface against infection, in need thereof, themethod comprising contacting the surface with an effective amount of oneor more preparations containing the iron complexes wherein thecomponents of the one or more preparations are coated onto the surfaceto be disinfected.

In some embodiments the one or more preparations may be applied to thesurface in the form of a spray, an aerosol, or a foam.

The coated surface may, for example, be formed on the surface of aninstrument selected from the group consisting of surgical instruments,cardiac and urinary catheters, implants, and ultrasound probes used insterile body cavities.

The coated surface may, for example, be formed on the surface of adevice selected from the group consisting of urinary catheter, stents,mucous extraction catheter, suction catheter, umbilical cannula, contactlenses, intrauterine devices, intravaginal and intraintestinal devices,endotracheal tubes, bronchoscopes, dental prostheses and orthodonticdevices, surgical instruments, dental instruments, tubing, dental waterlines, dental drain tubes, fabrics, paper, indicator strips (e.g., paperindicator strips or plastic indicator strips), adhesives (e.g., hydrogeladhesives, hot-melt adhesives, or solvent-based adhesives), bandages,tissue dressings or healing devices and occlusive patches, catheters,including central venous catheters, urinary catheters, dialysiscatheters, and indwelling catheters, cardiac implants, mechanical heartvalves, stents, ventricular assist devices, pacemakers, cardiac rhythmmanagement (CRM) devices, cardiac resynchronization therapy devices(CRTs), and implantable cardioverter defibrillators (ICDs), syntheticvascular grafts, arteriovascular shunts, cerebral spinal fluid shunts,cochlear devices, prosthetic joints, orthopedic implants, internalfixation devices, bone cements, percutaneous sutures, surgical mesh andsurgical patches including hernia repair mesh, breast reconstructionmesh, mesh for breast and face lifts, slings, and mesh for pelvic floorreconstruction, tracheal and ventilator tubing, wound dressings,biological implants, penile implants, intrauterine devices, endotrachealtubes, and contact lenses.

The coated surface may, for example, be formed on the surface of anarticle selected from the group consisting of an industrial pipeline,liquid distribution lines, oil and gas pipelines and cosmetic container.

The coated surface may, for example, be formed on the surface of, or beincorporated into, or onto, a household item, such as an item selectedfrom the group consisting of household disinfectants; laundry detergent;cleaning supplies; equipment involved in the leeching process or mining;wound care; toothpaste; mouth wash; dental floss; toothpicks; chewableproducts (including food products); a mouth shield; a dental instrument;dentures; dental retainers; dental braces including plastic braces (suchas Invisalign); bristles of toothbrushes; dental prostheses andorthodontic devices; chewable non-food items, foods, or toys, such asdog bones and biscuits; a vacuum system; HVAC ((heating, ventilation andair conditioning)) systems; vacuum cleaner bags; paint covering; wallcoverings; window frames; doors; door frames; cooling towers;humidifiers; vacuum cleaners; filters such as a vacuum filter, ahumidifier filter, hot tub filter, or a swimming pool filter; toys;plastic bottles; water jugs; tap and water spout; washing machines;dishwashers; animal water dishes; bathroom tiles and fixtures; sinks;showers; shower heads; toilets; toilets lids; toilet seats; sealants andgrout; towels; TUPPERWARE®; dishes; cups; utensils such as forks,spoons, knives, and spatulas; bowls; food storage containers; beveragestorage containers; cutting boards; dish drying trays; garbage bags;sinks; fish ponds; swimming pools; swimming pool liners; swimming poolskimmer; pond liners; bird baths; garden hose; water sprinkling lines;planters; and hot tubs.

The coated surface may, for example, be formed on the surface of, orincorporated into, or onto, an article, device or apparatus used in therearing and/or transport of animals, such as a chicken, for example, ameat-type chicken such as broiler chicken, or an egg-laying chicken suchas a pullet or hen, or a breeder chicken, other poultry, such as aturkey, geese, quail or ducks, or livestock, such as cattle, sheep,goats or swine, alpaca, banteng, bison, camel, cat, deer, dog, donkey,gayal, guinea pig, horse, llama, mule, rabbit, reindeer, water buffalo,yak, although the skilled person will appreciate that other feeds foranimals, including zoo animals, captive animals, game animals, fish(include freshwater and saltwater fish, fan led fish, and ornamentalfish), other marine and aquatic animals, including shellfish such as,but not limited to, oysters, mussels, clams, shrimps, prawns, lobsters,crayfish, crabs, cuttlefish, octopus, and squid, domestic animals suchas cats and dogs, rodents (such as mice, rats, guinea pigs, hamsters),and horses, are also provided, as well as any other domestic, wild andfanned animal, including mammals, marine animals, amphibians, birds,reptiles, insects and other invertebrates. In some embodiments, thedevice or apparatus used in the rearing and/or transport of animals maybe selected from an article, device or apparatus that is for thedelivery and/or containment of animal feed and/or animal drinking water.

Also provided is a composition comprising one or more conjugatedcomponents of one or more preparations as defined above, and an articlecoated with one or more of the conjugated components of one or morepreparations, or with the composition.

In one embodiment, the structure of the conjugated components of one ormore preparations comprise hydroxyapatite or derivative thereof, and theconjugate is capable of anchoring, or is anchored to, a dental tissue.

For example, in a further embodiment, conjugated forms of the componentsof one or more preparations, such as those wherein the components of oneor more preparations are conjugated to hydroxyapatite may be applied totooth tissues, such as tooth enamel, dentin and pulp in order to preventdental caries and infection. In another embodiment, the components ofone or more preparations can be applied using photo-reactive chemistry,for example, using conjugated forms such as those shown in FIGS. 7A andB.

4. Industrial, Cosmetic and Consumer Applications

The compositions can be used to disinfect industrial surfaces, bypreventing and/or removing biofilm buildup on such surfaces. In thisembodiment, the formation of the biofilm may be prevented or inhibited,or a preformed biofilm may be removed by a method that comprisesapplying a composition comprising the one or more preparationscontaining the iron complexes onto a surface in need thereof, forexample as a spray, foam, gel, powders; dish or laundry detergents(liquid or solid), surface wax, glass cleaner, etc.

5. Additional Medical Applications

In a further embodiment, the preparations containing the iron complexesand compositions comprising one or more of the preparations, can be usedto treat any medical condition associated with biofilm formation as aresult of microorganisms including, but not limited to, gram-negativeand gram-positive bacteria, including Pseudomonas, H. pylori, E.feacalis, Campylobacter, E. coli, EPEC, UPEC and Staphylococcus.

III. Preparations and Compositions

A. Preparations

A preparation as defined herein is a preparation obtainable by a processincluding bringing a moiety such as a tyrosinate moiety into contactwith an Fe(III)-containing substance. The Fe(III)-containing substancemay be anhydrous or a hydrate substance. In preferred embodiments, theFe(III)-containing substance is a hydrate such as, but not limited to,iron (III) chloride hexahydrate.

In one option, the tyrosinate moiety is tyrosine (for example, tyrosinemay be added to the reaction mixture and converted to a tyrosinate ionthrough the action of a base) and it is brought into contact with theFe(III)-containing substance in the presence of a base.

In another option, the tyrosinate moiety is a tyrosine salt and it isbrought into contact with an Fe(III)-containing substance. In the optionin which the tyrosinate moiety is a tyrosine salt, it may not benecessary to provide a base for its reaction with the Fe(III)-containingsubstance. The tyrosine salt may also be fully or partially dissolved ina solution prior to being brought into contact with theFe(III)-containing substance.

The tyrosinate moiety may, for example, be selected from the groupconsisting of tyrosine (as noted above, for example, tyrosine may beadded to the reaction mixture and converted to a tyrosinate ion throughthe action of a base), a salt of tyrosine, an anion of tyrosine, adouble anion of tyrosine, a deprotonated tyrosine, mono-deprotonatedtyrosine, and a double-deprotonated tyrosine. Examples of tyrosine saltsthat may be used include, without limitation, a sodium salt of tyrosine,a potassium salt of tyrosine, a lithium salt of tyrosine, and/or anammonium salt of tyrosine.

In one embodiment of the process, the tyrosinate moiety thereof isprovided in combination with a first solvent (e.g. in the form of asolution, suspension or slurry) prior to the tyrosinate moiety beingbrought into contact with the Fe(III)-containing substance. Prior tobeing brought into contact with the tyrosinate moiety thereof, theFe(III)-containing substance may be provided either as a solid or in theform of a solution, suspension or slurry with a second solvent (whichsecond solvent may be the same as or different from the first solvent).Preferably the first and second solvents are the same.

Particular solvents that may be mentioned in respect of the first andsecond solvents include aprotic polar solvents (e.g. acetonitrile), andmore particularly polar protic solvents (e.g. water and C ₁₋₄ alkylalcohols), as well as mixtures of such solvents. Preferably the firstand second solvents are the same and consist of one or more polar proticsolvents.

The amount of solvent required will depend upon the amount ofFe(III)-containing substance and the amount of tyrosinate moiety to beused in the process. Preferably the total amount of the first and secondsolvents (e.g. water) used in the process is at least 300 mL.

The reaction between tyrosine and the Fe(III)-containing substance isfacilitated by the presence of the base. The base may aid indeprotonating the tyrosine, thereby forming a deprotonated tyrosinatemoiety, and increasing the solubility of the tyrosine (in an ionic form)in aqueous media. For example, the solubility of tyrosine is greatlyincreased in aqueous media in which the pH is raised to at least 8.5. Inone embodiment the base is added to the tyrosine or other tyrosinatemoiety thereof before the tyrosine or other tyrosinate moiety thereof isbrought into contact with the Fe(III)-containing substance. Accordingly,the reaction process can also include the step of preparing or providinga tyrosine salt, such as a sodium salt of tyrosine, and then bringingtogether the tyrosine salt and the Fe(III)-containing substance in thepresence of the base. Optionally, the salt may be a double salt oftyrosine. The salt may be in either a solid form, or in solution, at thetime of being brought into contact with the Fe(III)-containingsubstance.

Suitable bases that may be used in the processes described hereininclude organic and inorganic bases. For example, the base may be anamine (e.g. a primary, secondary or, particularly, tertiary amine, suchas triethylamine, trimethylamine or diethylisopropylamine), anitrogen-based heterocycle (e.g. N-methylmorpholine or pyridine), analkoxide (e.g. an alkali metal alkoxide, such as sodium ethoxide), ahydroxide salt (e.g. an ammonium or alkali metal hydroxide, such aslithium, sodium or potassium hydroxide) or, particularly, a carbonate orbicarbonate salt (e.g. an alkaline earth or, particularly, an alkalimetal carbonate or bicarbonate). Particular bases that may be mentionedin this respect include trimethylamine, LiOH, KOH and NaOH.

The amount of base employed can vary, depending upon factors such as theparticular base used, the identity of the solvent, the rate of reactionthat is desired, etc. However, in certain embodiments of the process,the amount of base employed may be, for example, three, two orpreferably one equivalents relative to the tyrosine.

When used herein, the term “aqueous media” refers to solutions ofsubstances in which the solvent system comprises water, and optionallyfurther comprises one or more other solvents, such as water-miscibleorganic solvents (e.g. a C₁₋₄ alkyl alcohol, such as ethanol,isopropanol or, particularly, methanol). Further, when pH values arereferred to herein, those values may be determined by methods known tothose skilled in the art (e.g. by potentiometric measurements using aworking and a reference electrode), for example at room temperature(such as 25° C.).

The mixture containing the tyrosine, or other tyrosinate moiety, andbase may be heated to an elevated temperature (e.g. to a temperature ofat least 50° C.) either before or after the addition of theFe(III)-containing substance. The mixture may be held at an elevatedtemperature for a sufficient period of time (e.g. for at least about 5minutes, e.g. at least about 15 minutes) to allow the base to react withthe tyrosine, or other tyrosinate moiety.

In one embodiment the tyrosine (or other tyrosinate moiety) used ispredominantly L-tyrosine (or predominantly an L-tyrosinate moiety). Forexample, the tyrosine or other tyrosinate moiety used in the processcomprises may be at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% (e.g. atleast 95%) by weight L-configuration.

In another embodiment, the molar ratio of the Fe(III)-containingsubstance to the tyrosine, or other tyrosinate moiety, in the reactionmixture is in the range of about 1:1 to 1:4, such as about 1:2 or,preferably, about 1:3. “About” in that context can optionally includeone or more values selected from ±50%, 40%, 30%, 20%, 10% 5% or less ofthe stated value.

In another embodiment, the Fe(III)-containing substance comprises anFe(III) complex, or salt or hydrate thereof, or an Fe(III) salt, or ahydrate thereof Particular Fe(III) complexes and salts that may bementioned in this respect include Fe(III) halides (e.g. FeCl₃ andFeBr₃), Fe(OH)₃, Fe₂(SO₄)₃, Fe(HSO₄)₃, Fe(NO₃)₃, Fe(acetylacetonate)₃(“Fe(acac)₃”), Fe(acetate)₃, Fe(lactate)₃ and Fe (III) phosphate. Forthe avoidance of doubt, salts and hydrated forms of these Fe(III)complexes and Fe(III) salts are also included (such as FeCl₃.6H₂O). Inpreferred embodiments, the Fe(III) complexes and Fe(III) salts are thehydrated forms.

The amount of Fe(III) used in the process relative to the amount oftyrosine, or other tyrosinate moiety, may vary. However, in preferredembodiments of the process, the process involves the use of about 3(e.g. from 0.1, 0.5. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) equivalents oftyrosine, or other tyrosinate moiety, relative to the Fe(III) employed.

The preparations used in the methods of the invention are obtainable bya process including bringing the tyrosinate moiety into contact with anFe(III)-containing substance, optionally in the presence of a base, asdiscussed above. In certain embodiments, the bringing into contact stepis performed over a period of about one to 8 hours. In some otherembodiments, the bringing into contact step is performed over a periodof about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours,or 8 hours. In certain other embodiments, the bringing into contact stepis performed over a period of less than 1 hour, preferably about 1-60minutes, more preferably 1-25 minutes, most preferably 1-10 minutes. Incertain embodiments, the bringing into contact step is performed over aperiod of about 5 minutes. Additional steps that may be involved in theprocesses include precipitation of a solid product, separation of thesolid product from the mixture (e.g. by filtration), and drying thesolid product. Further processing steps that may be involved includestirring, heating and/or cooling the reaction mixture. The use of theterms “consisting of” or “consisting essentially of” in the context ofthe process for obtaining the preparations is intended to refer toprocesses which may include any of the above-mentioned additional steps.However, the terms “consisting of” or “consisting essentially of” in thecontext of the process for obtaining the preparations refer to processeswhich typically do not involve any additional purification steps (e.g.such processes may not involve or include any purification steps whichmay separate one or more Fe(III)-containing products from anotherFe(III)-containing product).

Thus, in a further embodiment of the method for producing a preparationas used by the invention, the preparation is obtainable by a processwhich consists of, or consists essentially of, bringing the tyrosinatemoiety into contact with an Fe(III)-containing substance, optionally inthe presence of a base, and further optionally further including one ormore steps selected from the group consisting of stirring the mixture,heating the mixture, cooling the mixture, precipitating a solid product,separating a solid product from the mixture, and drying the product.

In certain embodiments of the method for producing the preparationcontains an effective amount of an Fe(III)-containing product, which isobtained by the process described herein. The Fe(III)-containing productincludes Fe(III) complex with a tyrosine or other tyrosinate moiety. Inpreferred embodiments, the preparation contains an effective amount ofan Fe(III)-containing product, such as an Fe(III) complexed with atyrosine or other tyrosinate moiety wherein at least some of theFe(III)-containing product present in the preparation has a ratio of 1:3of Fe(III): tyrosine or other tyrosinate moiety

In a particular embodiment, the processes disclosed herein do notinclude the step of isolating Fe(tyrosine)₃ from the reaction mixture.

The preparation produced by the reaction process described herein willtypically be a mixture of different substances, and may contain at leasttwo Fe(III)-containing substances. The at least two Fe(III)-containingsubstances may optionally include Fe (III) that is bound by the aminogroup, acid group, or phenolic group of tyrosine, or other tyrosinatemoiety, and can optionally include tyrosine, or other tyrosinate moiety,bound by two different Fe (III) atoms. Additional substances may also bepresent in the preparation, including one or more (including all) of thesubstances selected from the group consisting of tyrosine or othertyrosinate moiety (such as in the free base, or salt form) as well asby-products of the reaction (e.g. salts, such as NaCl). The identitiesof the by-products may vary depending on the identities of the startingmaterials used in the reaction (in particular, the identities of theFe(III)-containing substance and the base). In a preferred embodiment,the preparation obtainable by the process defined herein comprisesFe(OH)₃. Additionally, or alternatively, in a further preferredembodiment, the preparation obtainable by the process defined hereincomprises tyrosine or other tyrosinate moiety (e.g. in the free base orsalt form; optionally in addition to Fe(OH)₃). It may be preferable forthe amount of tyrosine, or other tyrosinate moiety, present (e.g., notcomplexed to iron and present in the free base or salt form) in thepreparation to be less than 5% (w/w), although it can be difficult tosolubilize tyrosine and therefore preparations may, in somecircumstances comprise less than 5% detectable tyrosine but may, inpractice, contain additional undetectable levels of tyrosine.

Optionally, the preparation may be obtainable by a process consisting ofdissolving L-tyrosine (or other L-tyrosinate moiety), and LiOH in water,heating the mixture, separately dissolving FeCl₃ in water, and addingthe FeCl₃ solution to the mixture after completion of the heating step.In a preferred embodiment, the preparation may be obtainable by aprocess consisting of dissolving L-tyrosine (30 mmol) and LiOH.H₂O (30mmol) in water (250 ml), heating the mixture to 70° C. for 20 min,separately dissolving FeCl₃ (10 mmol) in water (3-5 ml) and adding theFeCl₃ solution to the tyrosine/LiOH mixture after completion of theheating step.

In another optional embodiment, the preparation may be different fromthe preparation obtainable by the foregoing process. Accordingly, thepreparation may be obtainable by a process including bringing tyrosine,or other tyrosinate moiety (such as a tyrosine salt) thereof, intocontact with an Fe(III)-containing substance, optionally in the presenceof a base as hereinabove defined, provided that the process does notconsist of dissolving L-tyrosine (30 mmol) and LiOH.H₂O (30 mmol) inwater (250 ml), heating the mixture to 70° C. for 20 min, separatelydissolving FeCl₃ (10 mmol) in water (3-5 ml) and adding the FeCl₃solution to the tyrosine/LiOH mixture after completion of the heatingstep.

Preparations produced by the foregoing methods will preferably containan effective amount of a complex of L-tyrosine (or other L-tyrosinatemoiety) with Fe III (the complex is referred to herein as “Fe-Tyr”, alsoin places is also denoted “Fe—Y”), such as a compound having thefollowing structure:

Additionally, or alternatively, in a further embodiment, the preparationobtainable by the process described herein contains an effective amountof a complex of tyrosine (or other tyrosinate moiety) with Fe IIIwherein the tyrosine (or other tyrosinate moiety coordinates the ferricion via just the amino group of the tyrosine or other tyrosinate moiety.Additionally, or alternatively, in yet other embodiments, thepreparation obtainable by the process described herein contains aneffective amount of a complex of tyrosine (or other tyrosinate moiety)with Fe III wherein at least some of the complexes formed by the processare μ-oxo trimers. Iron carboxylates often form trimers, with atriply-bridging oxygen at the center. In yet another embodiment, thepreparation obtainable by the process described herein contains aneffective amount of a complex of tyrosine (or other tyrosinate moiety)with Fe III wherein the preparation includes μ-oxo trimeric complexes.

In a further embodiment, a preparation may be a preparation thatinhibits biofilm formation by bacteria as measured in a plastic beadassay (such as in accordance with a method as described in theExamples), wherein the bacteria is grown in a medium containing thepreparation to form a growth suspension of the bacteria at 0.0001 OD/ml,the growth suspension is allowed to grow with plastic coated UV beads(Lascells), and the beads are assayed after 24 hours for the presence ofbiofilm formation on the beads (by counting bacteria after release fromthe beads), and compared to a control group where the bacteria is notgrown in the presence of the preparation. Preferably the preparationinhibits the binding of the bacteria to the plastic coated beads at alevel of inhibition that is at, or at least, about 1%, 2%, 3%, 4%, morepreferably at, or at least, about 5%, even more preferably at, or atleast, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, 100% or more of the level of inhibition of the binding of thebacteria to the plastic coated UV beads by either a preparation preparedaccording to the method of Example 14, or a complex of quinic acid withFe III, in which Fe III is present at the same molar concentration. Inparticularly preferred embodiment, the bacteria can be Enterococcusfaecalis, Staphylococcus epidermidis, Staphylococcus aureus,Campylobacter jejuni, Pseudomonas aeruginosa, Uropathogenic Escherichiacoli, and Enteropathogenic Escherichia coli.

In a further embodiment, a preparation may be a preparation thatinhibits binding of Helicobacter pylori to human gastric tissue (forexample as determined by a method as described in Example 5) at a levelof inhibition that is at, or at least, about 1%, 2%, 3%, 4%, morepreferably at, or at least, about 5%, even more preferably at, or atleast, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, 100% or more of the level of inhibition of the binding of thebacteria to human gastric tissue by either a preparation preparedaccording to the method of Example 14, or a complex of quinic acid withFe III, in which Fe III is present at the same molar concentration asmeasured by counting the average number of bacteria bound to the tissue.

In a further embodiment, a preparation may be a preparation thatinhibits biofilm formation of a bacteria, but does not inhibitplanktonic growth of the bacteria (for example, as determined using amethod as described in Example 7), wherein the bacteria can be one ormore of the following: Enterococcus faecalis, Staphylococcusepidermidis, Staphylococcus aureus, Campylobacter jejuni, Pseudomonasaeruginosa, Uropathogenic Escherichia coli, and EnteropathogenicEscherichia coli. Preferably the one or more preparations inhibitbiofilm formation (for example, as measured by coverage rate in Example7), at a level that is at, or at least, about 1%, 2%, 3%, 4%, morepreferably at, or at least, about 5%, even more preferably at, or atleast, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, 100% or more of the level of biofilm inhibition by apreparation prepared according to the method of Example 14, or a complexof quinic acid with Fe III, in which Fe III is present at the same molarconcentration.

In a further embodiment, a preparation may be a preparation for thetreatment of cystic fibrosis. In one embodiment, the preparation may bedelivered using a nebulizer spray. In another embodiment, thepreparation may be delivered in liposomes for the treatment of patientswith cystic fibrosis.

In a further embodiment, a preparation may be a preparation thatprevents attachment of bacteria to a surface (for example, whendetermined in accordance with a method as described in Example 13), andthe prevention of attachment of bacteria to the surface is at a levelthat is at, or at least, about 1%, 2%, 3%, 4%, more preferably at, or atleast, about 5%, even more preferably at, or at least, about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or moreof the level of bacteria attachment by a preparation prepared accordingto the method of Example 14, or a complex of quinic acid with Fe III, inwhich Fe III is present at the same molar concentration, for example asmeasured by optical density. In particularly preferred embodiment, thebacteria can be Enterococcus faecalis, Staphylococcus epidermidis,Staphylococcus aureus, Campylobacter jejuni, Pseudomonas aeruginosa,Uropathogenic Escherichia coli, and Enteropathogenic Escherichia coli.

The preparations may comprise components which are hydrates, or salts ofhydrates. For example, the preparations may comprise be Fe-Tyr.xH₂O. Thecompounds may also be hydrates containing salts, for example hydrateswith bases such as ammonium hydroxide, lithium hydroxide, sodiumhydroxide or potassium hydroxide present.

In the case of preparations comprising compounds which are Fe IIIcomplexes comprising ligands bound to the iron centre, as describedabove, in one option not all ligands will be the same in thecompositions comprising the Fe III complex compounds. For example, inthe case that the preparation comprises FeTyr, then this may be formedby creating a complex from Fe III and a commercial source of tyrosine(Tyr), which may include low levels (typically, less than 10%, such asless than 5% or about 2.5%) of one or more further amino acids, such ascysteine (Cys) and/or phenylalanine (Phe), and so in one optionalembodiment, when the compound is FeTyr, then some of the compounds inthe composition may include one or more alternative amino acids (e.g.Cys and/or Phe) as ligands. The same applies mutatis mutandis to otherligands used in the preparation of Fe III complexes.

Therefore, for example, in a preparation comprising an Fe III complex asdescribed above, it may be that less than 100% of the Fe III ligands areidentical, although preferably at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of theligands in the composition are identical. In that context, in oneembodiment the term “identical” discriminates between enantiomeric formsof ligand, that is, different enantiomers are not identical; whereas, inanother embodiment, the term “identical” can be applied to differentenantiomeric forms of ligand, that is, optionally different enantiomericforms of the same ligand are considered to be identical.

1. Derivatives

Preparations may optionally comprise derivatives of the above mentionedcompounds. The term “derivative” does not mean that the derivative issynthesized from the parent compound either as a starting material orintermediate, although this may be the case. The term “derivative” caninclude salts (for example, pharmaceutically acceptable salts),prodrugs, or metabolites of the parent compound. Derivatives includecompounds in which free amino groups in the parent compound have beenderivatized to form amine hydrochlorides, p-toluene sulfoamides,benzoxycarboamides, t-butyloxycarboamides, thiourethane-typederivatives, trifluoroacetylamides, chloroacetylamides, or formamides.Derivatives include compounds having one or more amino substituents orhydrogen groups replaced with substituted or unsubstituted alkyl,aminoalkyl, aryl, or heteroaryl groups having from 1 to 30 carbon atoms.

2. Salts

Preparations may optionally comprise the above-mentioned compounds inthe form of a salt, for example, a pharmaceutically acceptable salt.Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; and alkali or organic salts of acidic residues such ascarboxylic acids. The pharmaceutically acceptable salts include theconventional non-toxic salts or the quaternary ammonium salts of theparent compound formed, for example, from non-toxic inorganic or organicacids and inorganic or organic bases. Such conventional non-toxic saltsinclude those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acids; and thesalts prepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, tolunesulfonic,naphthalenesulfonic, methanesulfonic, ethane disulfonic, oxalic, andisethionic salts, and bases such as lithium hydroxide, sodium hydroxide,potassium hydroxide and ammonium hydroxide.

The pharmaceutically acceptable salts of the compounds can besynthesized from the parent compound, which contains a basic or acidicmoiety, by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins,Baltimore, Md., 2000, p. 704; and “Handbook of Pharmaceutical Salts:Properties, Selection, and Use,” P. Heinrich Stahl and Camille G.Wennuth, Eds., Wiley-VCH, Weinheim, 2002.

B. Antimicrobial Agents

Antimicrobial agents that may be used therapeutically and/ornon-therapeutically with the preparations, for example for the treatmentor prophylaxis of microbial infection, include, but are not limited to:(i) Aminoglycosides, including amikacin, gentamicin, kanamycin,neomycin, netilmicin, tobramycin, paromomycin, streptomycin,spectinomycin; (ii) Ansaycins, including geldanamycin, herbimycin,rifaximin, (iii) Carbacephem, including loracarbef, (iv) Carbapenems,including ertapenem, doripenem, imipenem/cilastatin, meropenem, (v)Cephalosporins, including cefadroxil, cefazolin, cefalotin orcefalothin, cephalexin, cefaclor, cefamandole, cefoxitin, cefprozil,cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime,cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone,cefepime, ceftaroline fosamil, ceftobiprole, (vi) Glycopeptides,including teicoplanin, vancomycin, telavancin, dalbavancin, oritavancin,(vii) Lincosamides, including clindamycin, lincomycin, (viii)Lipopeptides including daptomycin, (ix) Macrolides includingazithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, troleandomycin, telithromycin, spiramyin, (x)Monobactams, including aztreonam, (xi) Nitrofurans, includingfurazolidone, nitrofurantoin, (xii) Oxazolidinones, including linezolid,posizolid, radezolid, torezolid, (xiii) Penicillins, includingamoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin,dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin,oxacillin, penicillin G, penicillin V, piperacillin, temocillin,ticarcillin, amoxicillin/clavulanate, ampicillin/sulbactam,peperacillin/tazobactam, ticarcillin/clavulanate (xiv) Polypeptidesincluding bacitracin, colistin, polymyxin B, (xv)Quinolones/Fluoroquinolone, including ciprofloxacin, enoxacin,gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin,nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin,sparfloxacin, temafloxacin, (xvi) Sulfonamides, including mafenide,sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine,sulfamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine,sulfisoxazole, trimethoprim-sulfamethoxazole(co-trimoxazaole),sulfonamidochrysoidine, (xvii) Tetracyclines, including demeclocycline,doxycycline, minocycline, oxytetracycline, tetracycline, (xviii)clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide,isoniazid, pyrazinamide, rifampicin (rifampin), rifabutin, rifapentine,streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid,metronidazole, mupirocin, platensimycin, quinupristirildalfopristin,thiamphenicol, tigecycline, tinidazole, and trimethoprim; andcombinations thereof. The preparation may also be combined withtriclosan and chlorhexidine. Other antimicrobial agents include:aztreonam; cefotetan and its disodium salt; loracarbef; cefoxitin andits sodium salt; cefazolin and its sodium salt; cefaclor; ceftibuten andits sodium salt; ceftizoxime; ceftizoxime sodium salt; cefoperazone andits sodium salt; cefuroxime and its sodium salt; cefuroxime axetil;cefprozil; ceftazidime; cefotaxime and its sodium salt; cefadroxil;ceftazidime and its sodium salt; cephalexin; cefamandole nafate;cefepime and its hydrochloride, sulfate, and phosphate salt; cefdinirand its sodium salt; ceftriaxone and its sodium salt; cefixime and itssodium salt; cefpodoxime proxetil; meropenem and its sodium salt;imipenem and its sodium salt; cilastatin and its sodium salt;azithromycin; clarithromycin; dirithromycin; erythromycin andhydrochloride, sulfate, or phosphate salts, ethylsuccinate, and stearateforms thereof clindamycin; clindamycin hydrochloride, sulfate, orphosphate salt; lincomycin and hydrochloride, sulfate, or phosphate saltthereof, tobramycin and its hydrochloride, sulfate, or phosphate salt;streptomycin and its hydrochloride, sulfate, or phosphate salt; neomycinand its hydrochloride, sulfate, or phosphate salt; acetyl sulfisoxazole;colistimethate and its sodium salt; quinupristin; dalfopristin;amoxicillin; ampicillin and its sodium salt; clavulanic acid and itssodium or potassium salt; penicillin G; penicillin G benzathine, orprocaine salt; penicillin G sodium or potassium salt; carbenicillin andits disodium or indanyl disodium salt; piperacillin and its sodium salt;ticarcillin and its disodium salt; sulbactam and its sodium salt;moxifloxacin; ciprofloxacin; ofloxacin; levofloxacins; norfloxacin;gatifloxacin; trovafloxacin mesylate; alatrofloxacin mesylate;trimethoprim; sulfamethoxazole; demeclocycline and its hydrochloride,sulfate, or phosphate salt; doxycycline and its hydrochloride, sulfate,or phosphate salt; oxytetracycline and its hydrochloride, sulfate, orphosphate salt; chlortetracycline and its hydrochloride, sulfate, orphosphate salt; metronidazole; dapsone; atovaquone; rifabutin;linezolide; polymyxin B and its hydrochloride, sulfate, or phosphatesalt; sulfacetamide and its sodium salt; clarithromycin; and silverions, salts, and complexes.

A complex of L-tyrosine with Fe III (Fe-Tyr)can be combined with any oneor more of the foregoing antibiotics, either formulated together in thesame composition for administration or presented in separatecompositions for use separately, simultaneously or sequentially.

C. Excipients and Carriers

The preparations as defined above can be formulated for use inaccordance with any aspect and may, for example, be formulated in a waythat is suitable for enteral, parenteral, topical, or pulmonaryadministration.

The preparations can be combined with one or more pharmaceuticallyacceptable carriers and/or excipients that are considered safe andeffective and may be administered to an individual without causingundesirable biological side effects or unwanted interactions.

The carrier can include all components present in the pharmaceuticalformulation other than the active ingredient or ingredients. Thepreparations are included in the formulation in an effective amount toachieve the effect of the aspects of the present invention, for examplein an amount that is effective to inhibit biofilm formation or reducebiofilin buildup. An effective amount of a preparations provided to asubject may be an amount that is enough to provide the required degreeof reduction of microbial colonization. This may depend on the type ofpreparation and/or the size of the animal.

In one embodiment an effective amount of the preparations may be anamount that is effective to deliver the preparations to the site atwhich action is required with an Fe (III) concentration that ranges from1 μm to 1 M, preferably greater than 10 μM, 20 μM, 30 μM, 40 μM, 50 μM,60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150μM, 160 μM, 170 μM, 180 μM, 190 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600μM, 700 μM, 800 μM, 900 μM, 1 mM, 10 mM, 50 mM, 100 mM, 500 mM, 900 mMor more. A suitable Fe (III) concentration may be within the range ofabout 1 μm to about 1 mM, or about 30 μM to about 0.5 mM, or about 60 μMto about 0.3 mM. These concentrations may particularly apply to theperformance of the invention in the context of the aspects of thepresent invention.

In a further embodiment an effective amount of the preparation may be0.3 to 32 mg/day/kg bodyweight of the subject such as a chicken. Inanother embodiment an effective concentration of the preparations may bean amount effective to provide an Fe (III) concentration between 0.001to 1 mM for use in coatings or devices, or solutions.

The preparations can also be formulated for use as a disinfectant, forexample, in a hospital environment or for industrial application.

1. Parenteral Formulations

The preparationsfor use in accordance with any of the aspects and may beformulated for parenteral administration.

Parenteral administration may include administration to a patientintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intravitreally, intratumorally,intramuscularly, subcutaneously, subconjunctivally, intravesicularly,intrapericardially, intraumbilically, by injection, and by infusion.

Parenteral formulations can be prepared as aqueous compositions usingtechniques known in the art. Typically, such compositions can beprepared as injectable formulations, for example, solutions orsuspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a reconstitution medium prior toinjection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water(o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, one or more polyols (e.g., glycerol, propyleneglycol, and liquid polyethylene glycol), oils, such as vegetable oils(e.g., peanut oil, corn oil, sesame oil, etc.), and combinationsthereof. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride.

Solutions and dispersions of the active compounds as the free acid orbase or pharmacologically acceptable salts thereof can be prepared inwater or another solvent or dispersing medium suitably mixed with one ormore pharmaceutically acceptable excipients including, but not limitedto, surfactants, dispersants, emulsifiers, pH modifying agents,viscosity modifying agents, and combination thereof

Suitable surfactants may be anionic, cationic, amphoteric or nonionicsurface-active agents. Suitable anionic surfactants include, but are notlimited to, those containing carboxylate, sulfonate and sulfate ions.Examples of anionic surfactants include sodium, potassium, ammonium ionsof long chain alkyl sulfonates and alkyl aryl sulfonates such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 mono laurate, polyoxyethylene monolaurate,polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether,polyoxyethylene tridecyl ether, polypropylene glycol butyl ether,Poloxamer® (triblock copolymer of polyoxyethylene, followed by a blockof polyoxypropylene, followed by a block of polyoxyethylene) 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-.beta.-alanine, sodium N-lauryl-β-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

The formulation can contain a preservative to prevent the growth ofmicroorganisms. Suitable preservatives include, but are not limited to,parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. Theformulation may also contain an antioxidant to prevent degradation ofthe active agent(s).

The formulation is typically buffered to a pH of 3-8 for parenteraladministration upon reconstitution. Suitable buffers include, but arenot limited to, phosphate buffers, acetate buffers, and citrate buffers.Where preparations comprise acidic compounds, they may be advantageouslyformulated with a buffer in order to achieve a suitable pH, particularlyin the context of preparing injectable formulation, includingformulations for intravenous injection.

Water-soluble polymers are often used in formulations for parenteraladministration. Suitable water-soluble polymers include, but are notlimited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, andpolyethylene glycol.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent ordispersion medium with one or more of the excipients listed above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium and therequired other ingredients from those listed above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof The powders can be prepared in such a manner that theparticles are porous in nature, which can increase dissolution of theparticles. Methods for making porous particles are well known in theart.

(a) Controlled Release Formulations

The parenteral formulations described herein comprising one or morepreparationsfor use in accordance with any of the aspects may beformulated for controlled release including immediate release, delayedrelease, extended release, pulsatile release, and combinations thereof.

1. Nano- and microparticles

For parenteral administration, the one or more preparationsfor use inaccordance with any of the aspects of the present invention, andoptional one or more additional active agents, can be incorporated intomicroparticles, nanoparticles, or combinations thereof that providecontrolled release of the one of more preparations, or one or morecomponents thereof, and/or one or more additional active agents. Inembodiments wherein the formulations contains two or more activecomponents, such as drugs, then they can be formulated for the same typeof controlled release (e.g., delayed, extended, immediate, or pulsatile)or they can be independently formulated for different types of release(e.g., immediate and delayed, immediate and extended, delayed andextended, delayed and pulsatile, etc.).

For example, the one or more preparations and/or one or more additionalactive agents can be incorporated into polymeric microparticles, whichprovide controlled release of the active agent(s). Release of the activeagent (s) is controlled by diffusion of the drug(s) out of themicroparticles and/or degradation of the polymeric particles byhydrolysis and/or enzymatic degradation. Suitable polymers includeethylcellulose and other natural or synthetic cellulose derivatives.

Polymers, which are slowly soluble and form a gel in an aqueousenvironment, such as hydroxypropyl methylcellulose or polyethyleneoxide, can also be suitable as materials for drug containingmicroparticles. Other polymers include, but are not limited to,polyanhydrides, poly(ester anhydrides), polyesters, such as polylactide(PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA),polydioxanone, poly-3-hydroxybutyrate (PHB) and copolymers thereof,poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactoneand copolymers thereof, polymers including, but not limited to, polymersof glycolic acid, lactic acid, 1,4-dioxanone, trimethylene carbonate,3-hydroxybutyric acid, 4-hydroxybutyrate, e-caprolactone, includingpolyglycolic acid, polylactic acid, polydioxanone, polycaprolactone,copolymers of glycolic and lactic acids, such as VICRYL® polymer, MAXON®and MONOCRYL® polymers, and including poly(lactide-co-caprolactones);poly(orthoesters); polyanhydrides; poly(phosphazenes);polyhydroxyalkanoates; synthetically or biologically preparedpolyesters; polycarbonates; tyrosine polycarbonates; polyamides(including synthetic and natural polyamides, polypeptides, andpoly(amino acids)); polyesteramides; poly(alkylene alkylates);polyethers (such as polyethylene glycol, PEG, and polyethylene oxide,PEO); polyvinyl pyrrolidones or PVP; polyurethanes; polyetheresters;polyacetals; polycyanoacrylates; poly(oxyethylene)/poly(oxypropylene)copolymers; polyacetals, polyketals; polyphosphates;(phosphorous-containing) polymers; polyphosphoesters; polyalkyleneoxalates; polyalkylene succinates; poly(maleic acids); silk (includingrecombinant silks and silk derivatives and analogs); chitin; chitosan;modified chitosan; biocompatible polysaccharides; hydrophilic or watersoluble polymers, such as polyethylene glycol, (PEG) or polyvinylpyrrolidone (PVP), with blocks of other biocompatible or biodegradablepolymers, for example, poly(lactide), poly(lactide-co-glycolide, orpolycaprolcatone and copolymers thereof, including random copolymers andblock copolymers thereof. and combinations thereof.

Alternatively, the active agent can be incorporated into microparticlesprepared from materials which are insoluble in aqueous solution orslowly soluble in aqueous solution, but are capable of degrading withinthe GI tract by means including enzymatic degradation, surfactant actionof bile acids, and/or mechanical erosion. As used herein, the term“slowly soluble in water” refers to materials that are not dissolved inwater within a period of 30 minutes. Preferred examples include fats,fatty substances, waxes, wax-like substances and mixtures thereof.Suitable fats and fatty substances include fatty alcohols (such aslauryl, myristyl stearyl, cetyl or cetostearyl alcohol), fatty acids andderivatives, including but not limited to fatty acid esters, fatty acidglycerides (mono-, di- and tri-glycerides), and hydrogenated fats.Specific examples include, but are not limited to hydrogenated vegetableoil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenatedoils available under the trade name STEROTEX®, stearic acid, cocoabutter, and stearyl alcohol. Suitable waxes and wax-like materialsinclude natural or synthetic waxes, hydrocarbons, and normal waxes.Specific examples of waxes include beeswax, glycowax, castor wax,carnauba wax, paraffins and candelilla wax. As used herein, a wax-likematerial is defined as any material, which is noimally solid at roomtemperature and has a melting point of from about 30 to 300° C.

In some cases, it may be desirable to alter the rate of waterpenetration into the microparticles. To this end, rate-controlling(wicking) agents can be formulated along with the fats or waxes listedabove. Examples of rate-controlling materials include certain starchderivatives (e.g., waxy maltodextrin and drum dried corn starch),cellulose derivatives (e.g., hydroxypropylmethyl-cellulose,hydroxypropylcellulose, methylcellulose, and carboxymethyl-cellulose),alginic acid, lactose and talc. Additionally, a pharmaceuticallyacceptable surfactant (for example, lecithin) may be added to facilitatethe degradation of such microparticles.

Proteins, which are water insoluble, such as zein, can also be used asmaterials for the formation of active agent containing microparticles.Additionally, proteins, polysaccharides and combinations thereof, whichare water-soluble, can be formulated with drug into microparticles andsubsequently cross-linked to form an insoluble network. For example,cyclodextrins can be complexed with individual drug molecules andsubsequently cross-linked.

2. Method of Making Nano- and Microparticles

Encapsulation or incorporation of active agent, such as the one or morepreparationsfor use in accordance with any of the aspects of the presentinvention, into carrier materials to produce drug-containingmicroparticles can be achieved through known pharmaceutical formulationtechniques. In the case of formulation in fats, waxes or wax-likematerials, the carrier material is typically heated above its meltingtemperature and the active agent is added to form a mixture comprisingactive agent particles suspended in the carrier material, active agentdissolved in the carrier material, or a mixture thereof Microparticlescan be subsequently formulated through several methods including, butnot limited to, the processes of congealing, extrusion, spray chillingor aqueous dispersion. In a preferred process, wax is heated above itsmelting temperature, active agent is added, and the molten wax-drugmixture is congealed under constant stirring as the mixture cools.Alternatively, the molten wax-drug mixture can be extruded andspheronized to form pellets or beads. These processes are known in theart.

For some carrier materials it may be desirable to use a solventevaporation technique to produce active agent-containing microparticles.In this case active agent and carrier material are co-dissolved in amutual solvent and microparticles can subsequently be produced byseveral techniques including, but not limited to, forming an emulsion inwater or other appropriate media, spray drying or by evaporating off thesolvent from the bulk solution and milling the resulting material.

In some embodiments, active agent in a particulate form is homogeneouslydispersed in a water-insoluble or slowly water soluble material. Tominimize the size of the active agent particles within the composition,the active agent powder itself may be milled to generate fine particlesprior to formulation. The process of jet milling, known in thepharmaceutical art, can be used for this purpose. In some embodimentsactive agent in a particulate form is homogeneously dispersed in a waxor wax like substance by heating the wax or wax like substance above itsmelting point and adding the active agent particles while stirring themixture. In this case a pharmaceutically acceptable surfactant may beadded to the mixture to facilitate the dispersion of the active agentparticles.

The particles can also be coated with one or more modified releasecoatings. Solid esters of fatty acids, which are hydrolyzed by lipases,can be spray coated onto microparticles or active agent particles. Zeinis an example of a naturally water-insoluble protein. It can be coatedonto active agent containing microparticles or active agent particles byspray coating or by wet granulation techniques. In addition to naturallywater-insoluble materials, some substrates of digestive enzymes can betreated with cross-linking procedures, resulting in the formation ofnon-soluble networks. Many methods of cross-linking proteins, initiatedby both chemical and physical means, have been reported. One of the mostcommon methods to obtain cross-linking is the use of chemicalcross-linking agents. Examples of chemical cross-linking agents includealdehydes (gluteraldehyde and formaldehyde), epoxy compounds,carbodiimides, and genipin. In addition to these cross-linking agents,oxidized and native sugars have been used to cross-link gelatin.Cross-linking can also be accomplished using enzymatic means; forexample, transglutaminase has been approved as a GRAS substance forcross-linking seafood products. Finally, cross-linking can be initiatedby physical means such as thermal treatment, UV irradiation and gammairradiation.

To produce a coating layer of cross-linked protein surrounding activeagent containing microparticles or active agent particles, awater-soluble protein can be spray coated onto the microparticles andsubsequently cross-linked by the one of the methods described above.Alternatively, active agent-containing microparticles can bemicroencapsulated within protein by coacervation-phase separation (forexample, by the addition of salts) and subsequently cross-linked. Somesuitable proteins for this purpose include gelatin, albumin, casein, andgluten.

Polysaccharides can also be cross-linked to form a water-insolublenetwork. For many polysaccharides, this can be accomplished by reactionwith calcium salts or multivalent cations, which cross-link the mainpolymer chains. Pectin, alginate, dextran, amylose and guar gum aresubject to cross-linking in the presence of multivalent cations.Complexes between oppositely charged polysaccharides can also be formed;pectin and chitosan, for example, can be complexed via electrostaticinteractions.

(b) Injectable/Implantable Formulations

The one or more preparationsfor use in accordance with any of theaspects can be incorporated into injectable/implantable solid orsemi-solid implants, such as polymeric implants. In one embodiment, theone or more preparations are incorporated into a polymer that is aliquid or paste at room temperature, but upon contact with aqueousmedium, such as physiological fluids, exhibits an increase in viscosityto form a semi-solid or solid material. Exemplary polymers include, butare not limited to, hydroxyalkanoic acid polyesters derived from thecopolymerization of at least one unsaturated hydroxy fatty acidcopolymerized with hydroxyalkanoic acids. The polymer can be melted,mixed with the active substance and cast or injection molded into adevice. Such melt fabrication require polymers having a melting pointthat is below the temperature at which the substance to be delivered andpolymer degrade or become reactive. The device can also be prepared bysolvent casting where the polymer is dissolved in a solvent and the drugdissolved or dispersed in the polymer solution and the solvent is thenevaporated. Solvent processes require that the polymer be soluble inorganic solvents. Another method is compression molding of a mixedpowder of the polymer and the drug or polymer particles loaded with theactive agent.

Alternatively, the one or more preparations can be incorporated into apolymer matrix and molded, compressed, or extruded into a device that isa solid at room temperature. For example, the preparations can beincorporated into a biodegradable polymer, such as polyanhydrides,polyhydroalkanoic acids (PHAs), PLA, PGA, PLGA, polycaprolactone,polyesters, polyamides, polyorthoesters, polyphosphazenes, proteins andpolysaccharides such as collagen, hyaluronic acid, albumin and gelatin,and combinations thereof and compressed into solid device, such asdisks, or extruded into a device, such as rods. Further alternativepolymers for use in this context include polymers include, but are notlimited to, polymers of glycolic acid, lactic acid, 1,4-dioxanone,trimethylene carbonate, 3-hydroxybutyric acid, 4-hydroxybutyrate,e-caprolactone, including polyglycolic acid, polylactic acid,polydioxanone, polycaprolactone, copolymers of glycolic and lacticacids, such as VICRYL® polymer, MAXON® and MONOCRYL® polymers, andincluding poly(lactide-co-caprolactones); poly(orthoesters);polyanhydrides; poly(phosphazenes); polyhydroxyalkanoates; syntheticallyor biologically prepared polyesters; polycarbonates; tyrosinepolycarbonates; polyamides (including synthetic and natural polyamides,polypeptides, and poly(amino acids)); polyesteramides; poly(alkylenealkylates); polyethers (such as polyethylene glycol, PEG, andpolyethylene oxide, PEO); polyvinyl pyrrolidones or PVP; polyurethanes;polyetheresters; polyacetals; polycyanoacrylates;poly(oxyethylene)/poly(oxypropylene) copolymers; polyacetals,polyketals; polyphosphates; (phosphorous-containing) polymers;polyphosphoesters; polyalkylene oxalates; polyalkylene succinates;poly(maleic acids); silk (including recombinant silks and silkderivatives and analogs); chitin; chitosan; modified chitosan;biocompatible polysaccharides; hydrophilic or water soluble polymers,such as polyethylene glycol, (PEG) or polyvinyl pyrrolidone (PVP), withblocks of other biocompatible or biodegradable polymers, for example,poly(lactide), poly(lactide-co-glycolide, or polycaprolcatone andcopolymers thereof, including random copolymers and block copolymersthereof.

The release of the one or more preparations, or one or more componentsthereof, from the implant can be varied by selection of the polymer, themolecular weight of the polymer, and/or modification of the polymer toincrease degradation, such as the formation of pores and/orincorporation of hydrolyzable linkages. Methods for modifying theproperties of biodegradable polymers to vary the release profile of thepreparations, or one or more components thereof, from the implant arewell known in the art.

2. Enteral Formulations

The preparationsfor use in accordance with any of the aspects may beformulated for enteral administration.

Suitable oral dosage forms include tablets, capsules, solutions,suspensions, syrups, and lozenges. Tablets can be made using compressionor molding techniques well known in the art. Gelatin or non-gelatincapsules can be prepared as hard or soft capsule shells, which canencapsulate liquid, solid, and semi-solid fill materials, usingtechniques well known in the art.

Formulations may be prepared using a pharmaceutically acceptablecarrier. As generally used herein “carrier” includes, but is not limitedto, diluents, preservatives, binders, lubricants, disintegrators,swelling agents, fillers, stabilizers, and combinations thereof.

Carrier also includes all components of the coating composition, whichmay include plasticizers, pigments, colorants, stabilizing agents, andglidants.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name EUDRAGIT®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

“Diluents”, also referred to as “fillers,” are typically necessary toincrease the bulk of a solid dosage form so that a practical size isprovided for compression of tablets or formation of beads and granules.Suitable diluents include, but are not limited to, dicalcium phosphatedihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol,cellulose, microcrystalline cellulose, kaolin, sodium chloride, drystarch, hydrolyzed starches, pregelatinized starch, silicone dioxide,titanium oxide, magnesium aluminum silicate and powdered sugar.

“Binders” are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

“Lubricants” are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

“Disintegrants” are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (POLYPLASDONE® XL from GAF ChemicalCorp).

“Stabilizers” are used to inhibit or retard drug decompositionreactions, which include, by way of example, oxidative reactions.Suitable stabilizers include, but are not limited to, antioxidants,butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters;Vitamin E, tocopherol and its salts; sulfites such as sodiummetabisulphite; cysteine and its derivatives; citric acid; propylgallate, and butylated hydroxyanisole (BHA).

(a) Controlled Release Enteral Formulations

Oral dosage forms, such as capsules, tablets, solutions, andsuspensions, can be formulated for controlled release, for example, forthe controlled release of the one or more preparationsfor use inaccordance with any of the aspects of the present invention. Forexample, the one or more preparations and optional one or moreadditional active agents can be formulated into nanoparticles,microparticles, and combinations thereof, and encapsulated in a soft orhard gelatin or non-gelatin capsule or dispersed in a dispersing mediumto form an oral suspension or syrup. The particles can be formed of theactive agent and a controlled release polymer or matrix. Alternatively,the active agent particles can be coated with one or more controlledrelease coatings prior to incorporation in to the finished dosage form.

In another embodiment, the one or more preparations and optional one ormore additional active agents are dispersed in a matrix material, whichgels or emulsifies upon contact with an aqueous medium, such asphysiological fluids. In the case of gels, the matrix swells entrappingthe active agents, which are released slowly over time by diffusionand/or degradation of the matrix material. Such matrices can beformulated as tablets or as fill materials for hard and soft capsules.

In still another embodiment, the one or more preparations, and optionalone or more additional active agents are formulated into a sold oraldosage form, such as a tablet or capsule, and the solid dosage form iscoated with one or more controlled release coatings, such as a delayedrelease coatings or extended release coatings. The coating or coatingsmay also contain the one or more preparations and/or additional activeagents.

(1) Extended Release Dosage Forms

The extended release formulations are generally prepared as diffusion orosmotic systems, which are known in the art. A diffusion systemtypically consists of two types of devices, a reservoir and a matrix,and is well known and described in the art. The matrix devices aregenerally prepared by compressing the drug with a slowly dissolvingpolymer carrier into a tablet form. The three major types of materialsused in the preparation of matrix devices are insoluble plastics,hydrophilic polymers, and fatty compounds. Plastic matrices include, butare not limited to, methyl acrylate-methyl methacrylate, polyvinylchloride, and polyethylene. Hydrophilic polymers include, but are notlimited to, cellulosic polymers such as methyl and ethyl cellulose,hydroxyalkylcelluloses such as hydroxypropyl-cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, andCARBOPOL® 934 (cross-linked polyacrylate polymer), polyethylene oxidesand mixtures thereof. Fatty compounds include, but are not limited to,various waxes such as carnauba wax and glyceryl tristearate and wax-typesubstances including hydrogenated castor oil or hydrogenated vegetableoil, or mixtures thereof.

In certain preferred embodiments, the plastic material is apharmaceutically acceptable acrylic polymer, including but not limitedto, acrylic acid and methacrylic acid copolymers, methyl methacrylate,methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethylmethacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid),poly(methacrylic acid), methacrylic acid alkylamine copolymerpoly(methyl methacrylate), poly(methacrylic acid)(anhydride),polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), andglycidyl methacrylate copolymers.

In certain preferred embodiments, the acrylic polymer is comprised ofone or more ammonio methacrylate copolymers. Ammonio methacrylatecopolymers are well known in the art, and are described in NF XVII asfully polymerized copolymers of acrylic and methacrylic acid esters witha low content of quaternary ammonium groups.

In one preferred embodiment, the acrylic polymer is an acrylic resinlacquer such as that which is commercially available from Rohm Pharmaunder the tradename EUDRAGIT®. In further preferred embodiments, theacrylic polymer comprises a mixture of two acrylic resin lacquerscommercially available from Rohm Phauna under the trade names EUDRAGIT®RL30D and EUDRAGIT® RS30D, respectively. EUDRAGIT® RL30D and EUDRAGIT®RS30D are copolymers of acrylic and methacrylic esters with a lowcontent of quaternary ammonium groups, the molar ratio of ammoniumgroups to the remaining neutral (meth)acrylic esters being 1:20 inEUDRAGIT® RL30D and 1:40 in EUDRAGIT® RS30D. The mean molecular weightis about 150,000. EUDRAGIT® S-100 and EUDRAGIT® L-100 are alsopreferred. The code designations RL (high permeability) and RS (lowpermeability) refer to the permeability properties of these agents.EUDRAGIT® RL/RS mixtures are insoluble in water and in digestive fluids.However, multiparticulate systems formed to include the same areswellable and permeable in aqueous solutions and digestive fluids.

The polymers described above such as EUDRAGIT® RL/RS may be mixedtogether in any desired ratio in order to ultimately obtain asustained-release formulation having a desirable dissolution profile.Desirable sustained-release multiparticulate systems may be obtained,for instance, from 100% EUDRAGIT® RL, 50% EUDRAGIT® RL and 50% EUDRAGITt® RS, and 10% EUDRAGIT® RL and 90% EUDRAGIT® RS. One skilled in the artwill recognize that other acrylic polymers may also be used, such as,for example, EUDRAGIT® L.

Alternatively, extended release formulations can be prepared usingosmotic systems or by applying a semi-permeable coating to the dosageform. In the latter case, the desired drug release profile can beachieved by combining low permeable and high permeable coating materialsin suitable proportion.

The devices with different drug release mechanisms described above canbe combined in a final dosage form comprising single or multiple units.Examples of multiple units include, but are not limited to, multilayertablets and capsules containing tablets, beads, or granules. Animmediate release portion can be added to the extended release system bymeans of either applying an immediate release layer on top of theextended release core using a coating or compression process or in amultiple unit system such as a capsule containing extended and immediaterelease beads.

Extended release tablets containing hydrophilic polymers are prepared bytechniques commonly known in the art such as direct compression, wetgranulation, or dry granulation. Their formulations usually incorporatepolymers, diluents, binders, and lubricants as well as the activepharmaceutical ingredient. The usual diluents include inert powderedsubstances such as starches, powdered cellulose, especially crystallineand microcrystalline cellulose, sugars such as fructose, mannitol andsucrose, grain flours and similar edible powders. Typical diluentsinclude, for example, various types of starch, lactose, mannitol,kaolin, calcium phosphate or sulfate, inorganic salts such as sodiumchloride and powdered sugar. Powdered cellulose derivatives are alsouseful. Typical tablet binders include substances such as starch,gelatin and sugars such as lactose, fructose, and glucose. Natural andsynthetic gums, including acacia, alginates, methylcellulose, andpolyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilicpolymers, ethylcellulose and waxes can also serve as binders. Alubricant is necessary in a tablet formulation to prevent the tablet andpunches from sticking in the die. The lubricant is chosen from suchslippery solids as talc, magnesium and calcium stearate, stearic acidand hydrogenated vegetable oils.

Extended release tablets containing wax materials are generally preparedusing methods known in the art such as a direct blend method, acongealing method, and an aqueous dispersion method. In the congealingmethod, the drug is mixed with a wax material and either spray-congealedor congealed and screened and processed.

(2) Delayed Release Dosage Forms

Delayed release formulations can be created by coating a solid dosageform with a polymer film, which is insoluble in the acidic environmentof the stomach, and soluble in the neutral environment of the smallintestine.

The delayed release dosage units can be prepared, for example, bycoating an active agent or an active agent-containing composition with aselected coating material. The active agent-containing composition maybe, e.g., a tablet for incorporation into a capsule, a tablet for use asan inner core in a “coated core” dosage form, or a plurality of activeagent-containing beads, particles or granules, for incorporation intoeither a tablet or capsule.

Preferred coating materials include bioerodible, gradually hydrolyzable,gradually water-soluble, and/or enzymatically degradable polymers, andmay be conventional “enteric” polymers. Enteric polymers, as will beappreciated by those skilled in the art, become soluble in the higher pHenvironment of the lower gastrointestinal tract or slowly erode as thedosage form passes through the gastrointestinal tract, whileenzymatically degradable polymers are degraded by bacterial enzymespresent in the lower gastrointestinal tract, particularly in the colon.Suitable coating materials for effecting delayed release include, butare not limited to, cellulosic polymers such as hydroxypropyl cellulose,hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methyl cellulose acetate succinate,hydroxypropylmethyl cellulose phthalate, methylcellulose, ethylcellulose, cellulose acetate, cellulose acetate phthalate, celluloseacetate trimellitate and carboxymethylcellulose sodium; acrylic acidpolymers and copolymers, preferably formed from acrylic acid,methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylateand/or ethyl methacrylate, and other methacrylic resins that arecommercially available under the tradename EUDRAGIT® (Rohm Phatma;Westerstadt, Germany), including EUDRAGIT® L30D-55 and L100-55 (solubleat pH 5.5 and above), EUDRAGIT® L-100 (soluble at pH 6.0 and above),EUDRAGIT® S (soluble at pH 7.0 and above, as a result of a higher degreeof esterification), and EUDRAGITS® NE, RL and RS (water-insolublepolymers having different degrees of pettneability and expandability);vinyl polymers and copolymers such as polyvinyl pyrrolidone, vinylacetate, vinylacetate phthalate, vinylacetate crotonic acid copolymer,and ethylene-vinyl acetate copolymer; enzymatically degradable polymerssuch as azo polymers, pectin, chitosan, amylose and guar gum; zein andshellac. Combinations of different coating materials may also be used.Multi-layer coatings using different polymers may also be applied.

The preferred coating weights for particular coating materials may bereadily determined by those skilled in the art by evaluating individualrelease profiles for tablets, beads and granules prepared with differentquantities of various coating materials. It is the combination ofmaterials, method and form of application that produce the desiredrelease characteristics, which one can determine only from the clinicalstudies.

The coating composition may include conventional additives, such asplasticizers, pigments, colorants, stabilizing agents, glidants, etc. Aplasticizer is normally present to reduce the fragility of the coating,and will generally represent about 3 wt. % to 50 wt. %”, or 10 wt % to50 wt. %, relative to the dry weight of the polymer. Examples of typicalplasticizers include polyethylene glycol, propylene glycol, triacetin,dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutylsebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate,castor oil and acetylated monoglycerides. A stabilizing agent ispreferably used to stabilize particles in the dispersion. Typicalstabilizing agents are nonionic emulsifiers such as sorbitan esters,polysorbates and polyvinylpyrrolidone. Glidants are recommended toreduce sticking effects during film formation and drying, and willgenerally represent approximately 25 wt. % to 100 wt. % of the polymerweight in the coating solution. One effective glidant is talc. Otherglidants such as magnesium stearate and glycerol monostearates may alsobe used. Pigments such as titanium dioxide may also be used. Smallquantities of an anti-foaming agent, such as a silicone (e.g.,simethicone), may also be added to the coating composition.

3. Topical Formulations

The preparationsfor use in accordance with any of the aspects and may beformulated for topical administration.

The formulations may contain the one or more preparations discussedabove, alone or in combination, in an effective amount to prevent orinhibit biofilm formation on a surface, or reduce the amount of biofilmon a surface being treated. 1000 colony forming units (cfu) ofCampylobacter are enough to infect a human and cause disease in a human.

Therefore, in one embodiment, an effective amount of the one or morepreparations as defined.A of this application is, or are, enough of thepreparation(s), alone, or in combination with other compounds, to reducethe number of cfu of Campylobacter or other microorganism of interest onthe surface being treated to a number that is unlikely to, or which willnot, cause infection in humans.

Suitable dosage forms for topical administration include creams,ointments, salves, sprays, gels, lotions, irrigants, and emulsions.

“Buffers” are used to control pH of a composition. Preferably, thebuffers buffer the composition from a pH of about 4 to a pH of about7.5, more preferably from a pH of about 4 to a pH of about 7, and mostpreferably from a pH of about 5 to a pH of about 7. In a preferredembodiment, the buffer is triethanolamine.

“Emollients” are an externally applied agent that softens or soothesskin and are generally known in the art and listed in compendia, such asthe “Handbook of Pharmaceutical Excipients”, 4^(th) Ed., PharmaceuticalPress, 2003. These include, without limitation, almond oil, castor oil,ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esterswax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycolpalmitostearate, glycerin, glycerin monostearate, glyceryl monooleate,isopropyl myristate, isopropyl palmitate, lanolin, lecithin, lightmineral oil, medium-chain triglycerides, mineral oil and lanolinalcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil,starch, stearyl alcohol, sunflower oil, xylitol and combinationsthereof. In one embodiment, the emollients are ethylhexylstearate andethylhexyl palmitate.

“Emulsifiers” are surface active substances which promote the suspensionof one liquid in another and promote the formation of a stable mixture,or emulsion, of oil and water. Common emulsifiers are: metallic soaps,certain animal and vegetable oils, and various polar compounds. Suitableemulsifiers include acacia, anionic emulsifying wax, calcium stearate,carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol,diethanolamine, ethylene glycol palmitostearate, glycerin monostearate,glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin,hydrous, lanolin alcohols, lecithin, medium-chain triglycerides,methylcellulose, mineral oil and lanolin alcohols, monobasic sodiumphosphate, monoethanolamine, nonionic emulsifying wax, oleic acid,poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylenecastor oil derivatives, polyoxyethylene sorbitan fatty acid esters,polyoxyethylene stearates, propylene glycol alginate, self-emulsifyingglyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate,sorbitan esters, stearic acid, sunflower oil, tragacanth,triethanolamine, xanthan gum and combinations thereof. In oneembodiment, the emulsifier is glycerol stearate.

“Penetration enhancers” are known in the art and include, but are notlimited to, fatty alcohols, fatty acid esters, fatty acids, fattyalcohol ethers, amino acids, phospholipids, lecithins, cholate salts,enzymes, amines and amides, complexing agents (liposomes, cyclodextrins,modified celluloses, and diimides), macrocyclics, such as macrocyliclactones, ketones, and anhydrides and cyclic ureas, surfactants,N-methyl pyrrolidones and derivatives thereof, DMSO and relatedcompounds, ionic compounds, azone and related compounds, and solvents,such as alcohols, ketones, amides, polyols (e.g., glycols). Examples ofthese classes are known in the art.

“Preservatives” can be used to prevent the growth of fungi andmicroorganisms. Suitable antifungal and antimicrobial agents include,but are not limited to, benzoic acid, butylparaben, ethyl paraben,methyl paraben, propylparaben, sodium benzoate, sodium propionate,benzalkonium chloride, benzethonium chloride, benzyl alcohol,cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,and thimerosal.

“Surfactants” are surface-active agents that lower surface tension andthereby increase the emulsifying, foaming, dispersing, spreading andwetting properties of a product. Suitable non-ionic surfactants includeemulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers,polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters,benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate,poloxamer, povidone and combinations thereof. In one embodiment, thenon-ionic surfactant is stearyl alcohol.

(a) Emulsions

An emulsion is a preparation of one liquid distributed in small globulesthroughout the body of a second liquid. In particular embodiments, thenon-miscible components of the emulsion include a lipophilic componentand an aqueous component. The dispersed liquid is the discontinuousphase, and the dispersion medium is the continuous phase. When oil isthe dispersed liquid and an aqueous solution is the continuous phase, itis known as an oil-in-water emulsion, whereas when water or aqueoussolution is the dispersed phase and oil or oleaginous substance is thecontinuous phase, it is known as a water-in-oil emulsion. Either or bothof the oil phase and the aqueous phase may contain one or moresurfactants, emulsifiers, emulsion stabilizers, buffers, and otherexcipients. Preferred excipients include surfactants, especiallynon-ionic surfactants; emulsifying agents, especially emulsifying waxes;and liquid non-volatile non-aqueous materials, particularly glycols suchas propylene glycol. The oil phase may contain other oilypharmaceutically approved excipients. For example, materials such ashydroxylated castor oil or sesame oil may be used in the oil phase assurfactants or emulsifiers.

The oil phase may consist at least in part of a propellant, such as anHFA propellant. Either or both of the oil phase and the aqueous phasemay contain one or more surfactants, emulsifiers, emulsion stabilizers,buffers, and other excipients. Preferred excipients include surfactants,especially non-ionic surfactants; emulsifying agents, especiallyemulsifying waxes; and liquid non-volatile non-aqueous materials,particularly glycols such as propylene glycol. The oil phase may containother oily pharmaceutically approved excipients. For example, materialssuch as hydroxylated castor oil or sesame oil may be used in the oilphase as surfactants or emulsifiers.

A sub-set of emulsions are the self-emulsifying systems. These deliverysystems are typically capsules (hard shell or soft shell) comprised ofthe preparation dispersed or dissolved in a mixture of surfactant(s) andlipophilic liquids such as oils or other water immiscible liquids. Whenthe capsule is exposed to an aqueous environment and the outer gelatinshell dissolves, contact between the aqueous medium and the capsulecontents instantly generates very small emulsion droplets. Thesetypically are in the size range of micelles or nanoparticles. No mixingforce is required to generate the emulsion as is typically the case inemulsion formulation processes.

(b) Lotions

A lotion can contain finely powdered substances that are insoluble inthe dispersion medium through the use of suspending agents anddispersing agents. Alternatively, lotions can have as the dispersedphase liquid substances that are immiscible with the vehicle and areusually dispersed by means of emulsifying agents or other suitablestabilizers. In one embodiment, the lotion is in the form of an emulsionhaving a viscosity of between 100 and 1000 centistokes. The fluidity oflotions permits rapid and uniform application over a wide surface area.Lotions are typically intended to dry on the skin leaving a thin coat oftheir medicinal components on the skin's surface.

(c) Creams

Creams may contain emulsifying agents and/or other stabilizing agents.In one embodiment, the formulation is in the form of a cream having aviscosity of greater than 1000 centistokes, typically in the range of20,000-50,000 centistokes. Creams are often time preferred overointments, as they are generally easier to spread and easier to remove.

The difference between a cream and a lotion is the viscosity, which isdependent on the amount/use of various oils and the percentage of waterused to prepare the formulations. Creams are typically thicker thanlotions, may have various uses and often one uses more variedoils/butters, depending upon the desired effect upon the skin. In acream formulation, the water-base percentage is about 60-75% and theoil-base is about 20-30% of the total, with the other percentages beingthe emulsifier agent, preservatives and additives for a total of 100%.

(d) Ointments

Examples of suitable ointment bases include hydrocarbon bases (e.g.,petrolatum, white petrolatum, yellow ointment, and mineral oil);absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin,and cold cream); water-removable bases (e.g., hydrophilic ointment), andwater-soluble bases (e.g., polyethylene glycol ointments). Pastestypically differ from ointments in that they contain a larger percentageof solids. Pastes are typically more absorptive and less greasy thanointments prepared with the same components.

(e) Gels

Gels are semisolid systems containing dispersions of small or largemolecules in a liquid vehicle that is rendered semisolid by the actionof a thickening agent or polymeric material dissolved or suspended inthe liquid vehicle. The liquid may include a lipophilic component, anaqueous component or both. Some emulsions may be gels or otherwiseinclude a gel component. Some gels, however, are not emulsions becausethey do not contain a homogenized blend of immiscible components.Suitable gelling agents include, but are not limited to, modifiedcelluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose;Carbopol homopolymers and copolymers; and combinations thereof. Suitablesolvents in the liquid vehicle include, but are not limited to, diglycolmonoethyl ether; alkylene glycols, such as propylene glycol; dimethylisosorbide; alcohols, such as isopropyl alcohol and ethanol. Thesolvents are typically selected for their ability to dissolve thepreparation, or one or more components thereof. Other additives, whichimprove the skin feel and/or emolliency of the formulation, may also beincorporated. Examples of such additives include, but are not limitedto, isopropyl myristate, ethyl acetate, C₁₂-C₁₅ alkyl benzoates, mineraloil, squalane, cyclomethicone, capric/caprylic triglycerides, andcombinations thereof.

Foams

Foams consist of an emulsion in combination with a gaseous propellant.The gaseous propellant consists primarily of hydrofluoroalkanes (HFAs).Suitable propellants include HFAs such as 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures andadmixtures of these and other HFAs that are currently approved or maybecome approved for medical use are suitable. The propellants preferablyare not hydrocarbon propellant gases, which can produce flammable orexplosive vapors during spraying. Furthermore, the compositionspreferably contain no volatile alcohols, which can produce flammable orexplosive vapors during use.

4. Disinfecting and Cleaning Formulations

The preparationsfor use in accordance with any of the aspects may beformulated into cleaning formulations.

The cleaning formulations include formulations that are highlyefficacious for household cleaning applications (e.g., hard surfaceslike floors, countertops, tubs, tile, dishes and softer cloth materialslike clothing, sponges, paper towels, etc.), personal care applications(e.g. lotions, shower gels, soaps, shampoos, sprays, wipes, toothpaste,acne treatments, skin cleansers, mouthwash, wound irrigation solutions,towelettes, contact lenses and lens cases) and industrial and hospitalapplications (e.g., antifouling coatings, and disinfection ofinstruments, medical devices, gloves, filters, membranes, tubing,drains, pipes including gas pipes, oil pipes, drilling pipes, frackingpipes, sewage pipes, drainage pipes, hoses, animal carcasses, fishtanks, showers, children's toys, boat hulls, and cooling towers). Theseformulations are efficacious for cleaning surfaces which are infected orcontaminated with biofilm or for preventing the formation of biofilm onthese surfaces.

The preparations can be formulated into a solution in a suitable solventfor administration in a spray bottle, the preparations can be formulatedas an aerosol, as a foam, suitable for spraying onto surfaces, or, theycan be imbibed into a cloth or other item suitable for wiping down asurface to be disinfected. Methods for making formulations for use as adisinfectant in the forms are known in the art.

One embodiment provides the preparations in a composition containing apH dye indicator and an alkaline substance. The pH indicator dyeindicates what surface has been disinfected and ensures that asufficient time has passed to disinfect the surface. See for example,U.S. Publication No. 20140057987, which is incorporated by reference inits entirety.

Cleaning formulations can include one or more of the preparations and anacceptable carrier. The carrier can be in a wide variety of forms. Forexample, the carrier may be an aqueous-based solution or cleanser, analcohol-based solution or gel or an emulsion carrier, including, but notlimited to, oil-in-water, water-in-oil, water-in-oil-in-water, andoil-in-water-in-silicone emulsions. The carrier solution containing thepreparation(s) can be applied directly to the surface to be treated ordelivered via a suitable substrate.

The cleaning formulations can be formulated for use on the skin. Inthese embodiments the one or more preparations can be formulated in adermatologically acceptable carrier. The dermatologically acceptablecarriers can also be, for example, formulated as alcohol or water basedhand cleansers, toilet bars, liquid soaps, shampoos, bath gels, hairconditioners, hair tonics, pastes, or mousses.

Cleaning formulations can contain one or more surfactants. Thesurfactant is suitably selected from anionic, nonionic, zwitterionic,amphoteric and ampholytic surfactants, as well as mixtures of thesesurfactants. Such surfactants are well known to those skilled in thedetergency art. Non limiting examples of possible surfactants includeisoceteth-20, sodium methyl cocoyl taurate, sodium methyl oleoyltaurate, and sodium lauryl sulfate. Examples of a broad variety ofadditional surfactants are described in McCutcheon's Detergents andEmulsifiers. North American Edition (1986), published by AlluredPublishing Corporation. The cleansing formulations can optionallycontain, at their art-established levels, other materials which areconventionally used in cleansing formulations.

Additional carriers suitable for the cleaning formulations may includevarious substrate-based products. In such instances, the presentformulations may be impregnated into or onto the substrate products andmay be allowed to remain wet or may be subjected to a drying process.For instance, suitable carriers include, but are not limited to, dry andwet wipes suitable for personal care and household use (e.g., nonwovenbaby wipes, household cleaning wipes, surgical preparation wipes, etc.);diapers; infant changing pads; dental floss; personal care and householdcare sponges or woven cloths (e.g., washcloths, towels, etc.);tissue-type products (e.g. facial tissue, paper towels, etc.); anddisposable garments (e.g., gloves, smocks, surgical masks, infant bibs,socks, shoe inserts, etc.). Cleaning formulations can be incorporatedinto various household care products including, but not limited to, hardsurface cleaners (e.g., disinfectant sprays, liquids, or powders); dishor laundry detergents (liquid or solid), floor waxes, glass cleaners,etc.

Exemplary carriers can include aqueous solutions, e.g. having from about0% to about 98.8%, by weight of the composition, of water. Additionally,carriers may contain an aqueous alcohol solution. The amount of alcoholpresent in the alcohol solution will vary depending on the type ofproduct in which the composition is incorporated, i.e. say a wipe wherethe preferred amount of alcohol present would be from about 0% to about25% whereas a hand sanitizer preferably contains from about 60% to about95%, of alcohol. Therefore, suitable deimatologically acceptable alcoholsolutions or gels may contain from about 0% to about 95%, by weight ofthe composition, of an alcohol.

Alcohols suitable for inclusion in the alcohol solutions of the carrierinclude, but are not limited to, monohydric alcohols, dihydric alcohols,and combinations thereof. More preferred alcohols are selected from thegroup consisting of monohydric linear or branched C₂-C₁₈ alcohols. Themost preferred alcohols are selected from the group consisting ofethanol, isopropanol, n-propanol, butanol, and combinations thereof. Thecleaning formulations which contain an alcohol solution may be anhydrousor water containing.

Thickeners can be added to the water or alcohol based to form a gel.Examples of suitable thickeners include, but are not limited to,naturally-occurring polymeric materials such as sodium alginate, xanthangum, quince seed extract, tragacanth gum, starch and the like,semi-synthetic polymeric materials such as cellulose ethers (e.g.hydroxyethyl cellulose, methyl cellulose, carboxymethyl cellulose,hydroxy propylmethyl cellulose), polyvinylpyrrolidone, polyvinylalcohol, guar gum, hydroxypropyl guar gum, soluble starch, cationiccelluloses, cationic guars and the like and synthetic polymericmaterials such as carboxyvinyl polymers, polyvinylpyrrolidone, polyvinylalcohol, polyacrylic acid polymers, polymethacrylic acid polymers,polyvinyl acetate polymers, polyvinyl chloride polymers, andpolyvinylidene chloride polymers. Inorganic thickeners may also be usedsuch as aluminum silicates, such as, for example, bentonites, or amixture of polyethylene glycol and polyethylene glycol stearate ordistearate.

The cleaning formulations can contain, in addition to the one or morepreparations as described above, one or more antimicrobial or antifungalagents. Such agents are capable of destroying microbes, preventing thedevelopment of microbes or preventing the pathogenic action of microbes.Examples of additional antimicrobial and antifungal agents includeβ-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin,tetracycline, erythromycin, amikacin, 2,4,4′-trichloro-2′-hydroxydiphenyl ether (TRICLOSAN®), phenoxyethanol, phenoxy propanol,phenoxyisopropanol, doxycycline, capreomycin, chlorhexidine,chlortetracycline, oxytetracycline, clindamycin, ethambutol, hexamidineisethionate, metronidazole, pentamidine, gentamicin, kanamycin,lineomycin, methacycline, methenamine, minocycline, neomycin,netilmicin, paromomycin, streptomycin, tobramycin, miconazole,tetracycline hydrochloride, erythromycin, zinc erythromycin,erythromycin estolate, erythromycin stearate, amikacin sulfate,doxycycline hydrochloride, capreomycin sulfate, chlorhexidine gluconate,chlorhexidine hydrochloride, chlortetracycline hydrochloride,oxytetracycline hydrochloride, clindamycin hydrochloride, ethambutolhydrochloride, metronidazole hydrochloride, pentamidine hydrochloride,gentamicin sulfate, kanamycin sulfate, lineomycin hydrochloride,methacycline hydrochloride, methenamine hippurate, methenaminemandelate, minocycline hydrochloride, neomycin sulfate, netilmicinsulfate, paromomycin sulfate, streptomycin sulfate, tobramycin sulfate,miconazole hydrochloride, ketaconazole, amanfadine hydrochloride,amanfadine sulfate, octopirox, parachlorometa xylenol, nystatin,tolnaftate, pyrithiones (especially zinc pyrithione which is also knownas ZPT), dimethyldimethylol hydantoin (GLYDANT®),methylchloroisothiazolinone/methylisothiazolinone (KATHON CG®), sodiumsulfite, sodium bisulfite, imidazolidinyl urea (Germall 115®),diazolidinyl urea (GERMAILL II®), benzyl alcohol,2-bromo-2-nitropropane-1,3-diol (BRONOPOL®), formalin (formaldehyde),iodopropenyl butylcarbamate (POLYPHASE P100®), chloroacetamide,methanamine, methyldibromonitrile glutaronitrile(1,2-Dibromo-2,4-dicyanobutane or TEKTAMER®), glutaraldehyde,5-bromo-5-nitro-1,3-dioxane (BRONIDOX®), phenethyl alcohol,o-phenylphenol/sodium o-phenylphenol, sodium hydroxymethylglycinate(SUTTOCIDE A®), polymethoxy bicyclic oxazolidine (NUOSEPt C®),dimethoxane, thimersal dichlorobenzyl alcohol, captan, chlφhenenesin,dichlorophene, chlorbutanol, glyceryl laurate, halogenated diphenylethers like 2,4,4′-trichloro-2′-hydroxy-diphenyl ether (TRICLOSAN® orTCS), 2,2′-dihydroxy-5,5′-dibromo-diphenyl ether, phenolic compoundslike phenol, 2-methyl phenol, 3-methyl phenol, 4-methyl phenol, 4-ethylphenol, 2,4-dimethyl phenol, 2,5-dimethyl pPhenol, 3,4-dimethyl phenol,2,6-dimethyl phenol, 4-n-propyl phenol, 4-n-butyl phenol, 4-n-amylphenol, 4-tert-amyl phenol, 4-n-hexyl phenol, 4-n-heptyl phenol, mono-and poly- alkyl and aromatic halophenols such as p-chlorophenol, methylp-chlorophenol, ethyl p-chlorophenol, n-propyl p-chlorophenol, n-butylp-chlorophenol, n-amyl p-chlorophenol, sec-amyl p-chlorophenol, n-hexylp-chlorophenol, cyclohexyl p-chlorophenol, n-heptyl p-chlorophenol,n-octyl p-chlorophenol, o-chlorophenol, methyl o-chlorophenol, ethylo-chlorophenol, n-propyl o-chlorophenol, n-butyl o-chlorophenol, n-amylo-chlorophenol, tert-amyl o-chlorophenol, n-hexyl o-chlorophenol,n-heptyl o-chlorophenol, o-benzyl p-chlorophenol, o-benzyl-m-methylp-chlorophenol, o-benzyl-m, m-dimethyl p-chlorophenol, o-phenylethylp-chlorophenol, o-phenylethyl-m-methyl p-chlorophenol, 3-methylp-chlorophenol, 3,5-dimethyl p-chlorophenol, 6-ethy 1-3 -methylp-chlorophenol, 6-n-propyl-3-methyl p-chlorophenol, 6-iso-propyl-3-methyl p-chlorophenol, 2-ethyl-3,5-dimethyl p-chlorophenol,6-sec-butyl-3 -methyl p-chlorophenol, 2-iso-propyl-3,5-dimethylp-chlorophenol, 6-diethylmethyl-3 -methyl p-chlorophenol,6-iso-propyl-2-ethyl-3 -methyl p-chlorophenol, 2-sec-amyl-3,5-dimethylp-chlorophenol, 2-diethylmethyl-3,5-dimethyl p-chlorophenol,6-sec-octyl-3-methyl p-chlorophenol, p-chloro-m-cresol, p-bromophenol,methyl p-bromophenol, ethyl p-bromophenol, n-propyl p-bromophenol,n-butyl p-bromophenol, n-amyl p-bromophenol, sec-amyl p-bromophenol,n-hexyl p-bromophenol, cyclohexyl p-bromophenol, o-bromophenol,tert-amyl o-bromophenol, n-hexyl o-bromophenol, n-propyl-m,m-dimethylo-bromophenol, 2-phenyl phenol, 4-chloro-2-methyl phenol, 4-chloro-3-methyl phenol, 4-chloro-3,5-dimethyl phenol,2,4-dichloro-3,5-dimethylphenol, 3,4,5,6-terabromo-2-methylphenol,5-methyl-2-pentylphenol, 4-isopropyl-3-methylphenol,para-chloro-meta-xylenol (PCMX), chlorothymol,5-chloro-2-hydroxydiphenylmethane, resorcinol and its derivativesincluding methyl resorcinol, ethyl resorcinol, n-propyl resorcinol,n-butyl resorcinol, n-amyl resorcinol, n-hexyl resorcinol, n-heptylresorcinol, n-octyl resorcinol, n-nonyl resorcinol, phenyl resorcinol,benzyl resorcinol, phenylethyl resorcinol, phenylpropyl resorcinol,p-chlorobenzyl resorcinol, 5-chloro 2,4-dihydroxydiphenyl methane,4′-chloro 2,4-dihydroxydiphenyl methane, 5-bromo 2,4-dihydroxydiphenylmethane, and 4′-bromo 2,4-dihydroxydiphenyl methane, bisphenoliccompounds like 2,2′-methylene his (4-chlorophenol), 2,2′-methylene bis(3,4,6-trichlorophenol), 2,2′-methylene bis (4-chloro-6-bromophenol),bis (2-hydroxy-3,5-dichlorophenyl) sulphide, and bis(2-hydroxy-5-chlorobenzyl)sulphide, benzoic esters (parabens) likemethylparaben, propylparaben, butylparaben, ethylparaben,isopropylparaben, isobutylparaben, benzylparaben, sodium methylparaben,and sodium propylparaben, halogenated carbanilides (e.g.,3,4,4′-trichlorocarbanilides (TRICLOCARBAN® or TCC),3-trifluoromethyl-4,4′-dichlorocarbanifide, 3,3′,4-trichlorocarbanilide,etc.), cationic actives such as benzalkonium chloride, and clotrimazole.Another class of antimicrobial agents (specifically antibacterialagents) which are useful, are the so-called “natural” antibacterialactives, referred to as natural essential oils. Typical naturalessential oil antibacterial actives include oils of anise, lemon,orange, rosemary, wintergreen, thyme, lavender, cloves, hops, tea tree,citronella, wheat, barley, lemongrass, cedar leaf, cedarwood, cinnamon,fleagrass, geranium, sandalwood, violet, cranberry, eucalyptus, vervain,peppermint, gum benzoin, basil, fennel, fir, balsam, menthol, ocmeaoriganum, Hydastis carradensis, Berberidaceae daceae, Ratanhiae andCurcuma longa.

The cleaning formulations may be packaged in a variety of suitablepackaging known to those skilled in the art. The liquid formulations maydesirably be packaged in manually operated spray dispensing containers,which are usually made of synthetic organic polymeric plastic materials.Accordingly, disinfecting formulations containing the one or morepreparations of the present invention, and packaged in a spraydispenser, preferably in a trigger spray dispenser or a pump spraydispenser, are envisioned. Spray-type dispensers allow to uniformlyapply to a relatively large area of a surface to be disinfected a liquidcleaning formulations described herein.

The preparations can be impregnated into a nonwoven absorbent wipe.Disinfectant wet wipes are also disclosed for example in U.S. Pat. No.8,563,017.

The preparations can be in an aqueous foam with a special surfactantsystem capable of generating a foam. See U.S. Pat. No. 8,097,265, U.S.Pat. No. 5,891,922 and U.S. Pat. No. 4,889,645.

The preparations can also be in a pressurized spray aerosol. See also,U.S. Publication No. 20010053333 which discloses a liquid flash-dryaerosol disinfectant composition with a flash vaporization component andan effective amount of an antimicrobial agent.

It is within the abilities of one of ordinary skill in the art todetermine the effective amount of the one or more preparations toinclude in an aerosol, foam, solution or disinfectant cloth for thepurpose of sterilizing for example, high risk hospital surfaces.

D. Conjugation and Immobilization of Preparations, or Components Thereof

The one or more components may be presented as conjugated and/orimmobilized compounds.

The one or more components of the one or more preparations may beconjugated with other agents in order to retain them on surfaces, forexample, to prevent biofilm formation on a surface. In one embodiment,the one or more components of the one or more preparations may beconjugated to an agent that has affinity for a surface in order toretain the compounds on that surface. For example, they may beconjugated to an agent wherein the agent is a polymer or oligomer, andthe polymer or oligomer has a high affinity for the surface.

In another embodiment the one or more components of the one or morepreparations may be conjugated to an agent wherein the agent comprises areactive moiety suitable for anchoring to a surface. The reactive moietymay, for example, be photo-reactive, or capable of coupling covalentlyto a surface. The reactive moiety may also incorporate spacers andlinkers and other functional groups in order to place the compound in adesired location relative to the surface. FIGS. 7A-C are examples of howFeQ (Fe-QA; ferric quinate) may be conjugated to an agent comprising areactive moiety suitable for anchoring to a surface. In each of thethree examples, FeQ is conjugated to a calix[4] arene frame thatcomprises a reactive moiety. In FIG. 7A, FeQ is conjugated via a linkerto a calix[4] arene frame that contains a photoreactive functionalgroup. FIG. 7B is a variant of FIG. 7A which shows that the reactivemoiety can be positioned at a different location on the calix[4] areneframe. FIG. 7C is an example of FeQ conjugated to a calix[4] areneframe, wherein the latter is functionalized with thiol groups that arecapable of reacting with surfaces. It should be understood thatdifferent linkers or no linkers may be used, and that other agents maybe used instead of the calix[4] arene frame, including cyclodextrins andother polymers and oligomers.

In yet another embodiment, the one or more components of the one or morepreparations may be conjugated to an agent that comprises a substancewith an affinity for a surface. The agent may incorporate spacers andlinkers and other functional groups in order to place the compound inthe desired location relative to the surface. In one embodiment, theagent contains hydroxyapatite.

E. Feeds and Feed Supplements

One or more of the preparations can be formulated into growth promotingformulations.

F. Treatment to Promote Growth

Preparations containing the iron complexes above, are particularlyuseful in promoting growth. The one or more preparations may be added toanimal feed or animal drinking water in order to promote growth.Addition of the one or more preparations to feed or drinking waterresults in improved growth. It has also been discovered that the one ormore preparations can be added to animal feed or animal drinking waterin order to decrease the mortality adjusted feed conversion ratio. Thusit is possible to use the one or more preparations to decrease theamount of feed necessary for an animal to grow. The one or morepreparations may further be administered with other animal additives,and may be administered in commercial feeds. In a preferred embodiment,the one or more preparations are administered in feeds.

It has also been discovered that the one or more preparations can beadministered to animals that are in a stressed environment in order toimprove their growth performance In a stressed environment the one ormore preparations promote growth that yields animals with higher averagebody weights. The one or more preparations also decrease mortalityadjusted feed conversion ratios in stressed environments.

EXAMPLES

The following non-limiting examples are included to demonstrateparticular embodiments of the various aspects of the present invention.In some instances, comparative examples are provided to show the effectof the preparations in comparison to preparations of Fe III complexeswith quinate (FeQ), with DOPA (FeDOPA) or with phenylalanine (FePhe). Itshould be appreciated by those of skill in the art that the techniquesdisclosed in the examples which follow represent techniques discoveredby the inventor to function well in the practice of the invention, andthus can be considered to constitute preferred modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 Inhibition of Biofilm Formation on Beads Surface byEnterococcus faecalis NCTC 12697 using Fe-QA

Materials and Methods

Bacteria (Enterococcus faecalis NCTC 12697, Staphylococcus epidermidisF1513 and Staphylococcus aureus ATCC 25923) were grown on Brain heartinfusion (BHI) passaged to new medium either containing Fe-QA or alone.Growth suspensions were prepared at 0.0001 OD/mi and then allowed togrow at 37° C. under nounal atmospheric conditions for 24 h in BHI withplastic coated UV beads (Lascells). After 48 h, 10 μl suspension wasserially diluted 10 fold to 10-3, 10-4, 10-5, 10-6, 10-7, 10-8. For eachdilution, 10 μl was spotted on BHI agar plates and colonies countedafter 24 h. The beads were also removed washed in PBS before adding to 1ml PBS. After vortex mixing, 10 μl of the cell suspension was seriallydiluted as above and cell counts carried out.

Results

Enterococcus faecalis causes many of the antibiotic resistant infectionsin hospitals, a consequence of its inherent resistance to certainantibiotics and of its ability to survive and proliferate in theintestinal tract. A Ser/Thr kinase in Enterococcus faecalis is found tomediate antimicrobial resistance. Studies have shown that PrkC, aone-component signaling protein containing a eukaryotic-type Ser/Thrkinase domain, allowed for inherent antimicrobial resistance andintestinal persistence of E. faecalis (Kristich, et al., Proc. Nat.Acad. Sci. USA, 104(9):3508-3513 (2007)). Kristich, et al. found that anE. faecalis mutant lacking PrkC grew at a wild-type rate in the absenceof antimicrobial stress but showed enhanced sensitivity tocell-envelope-active compounds, including antibiotics that targetedcell-wall biogenesis and bile detergents. PrkC regulates physiologicalprocesses in E. faecalis that are key to its success as a nosocomialpathogen.

The effect of Fe-QA on biofilm formation by E. faecalis was tested asdescribed in the materials and methods. The data (FIGS. 1A and 1B) showsthat Fe-QA inhibited E. faecalis biofilm formation as measured followingtreatment of E. faecalis grown on plastic coated UV beads.

Example 2 Inhibition of Biofilm Formation on Beads Surface byStaphylococcus epidermidis F1513 using Fe-QA

Materials and Methods

The effect of Fe-QA on biofilm formation by S. epidermidis F1513 wastested as described in the materials and methods of Example 1.

Results

The data (FIG. 2A and 2B) shows that Fe-QA inhibited S. epidermidisbiofilm formation as measured following treatment of S. epidermidisgrown on plastic coated UV beads.

Example 3 Inhibition of Biofilm Formation on Beads Surface byStaphylococcus aureus ATCC 25923 using Fe-QA

The treatment of choice for S. aureus infection is penicillin; in mostcountries, however, penicillin resistance is extremely common, andfirst-line therapy is most commonly a penicillinase-resistant β-lactamantibiotic (for example, oxacillin or flucloxacillin). Combinationtherapy with gentamicin may be used to treat serious infections, such asendocarditis, but its use is controversial because of the high risk ofdamage to the kidneys (Cosgrove, et al., Clin Infect Dis, 48(6):713-721(2009). The duration of treatment depends on the site of infection andon severity.

Materials and Methods

The effect of Fe-QA on biofilm formation by S. aureus was tested asdescribed in the materials and methods of Example 1.

Results

The data (FIG. 3A and 3B) shows that Fe-QA inhibited S. aureus biofilmformation as measured following treatment of S. aureus grown on plasticcoated UV beads.

Example 4 Fe-QA Prevention of Biofilm Formation by PseudomonasAeruginosa and Uropathogenic E. coli (UPEC).

Materials and Methods

Pseudomonas aeruginosa PAO-1, and a clinically isolated uropathogenicEscherichia coli UPEC-536 were routinely grown on either LB(Luria-Bertani, Oxoid, UK) agar plates at 37° C. or in broth at 37° C.with 200 rpm shaking UV-sterilized glass slides were incubated in either15 mL RPMI-1640 defined medium (Sigma, UK) or 15 mL RPMI-1640 with Fe-QAinoculated with diluted (OD₆₀₀=0.01) bacteria from overnight cultures at37° C. with 60 rpm shaking for 72 hours. The slides were removed frombacterial culture and washed with 15 mL phosphate buffered saline atroom temperature for 5 minutes three times and then rinsed withdistilled H₂O. After washing, the slides were stained with 20 μM SYTO17dye (Invitrogen, UK) at room temperature for 30 minutes. After removingexcess staining dye and air-drying, the samples were examined using aCarl Zeiss LSM 700 Laser Scanning Microscope with ZEN 2009 imagingsoftware (Carl Zeiss, Germany). The coverage rate of bacteria on thesurface was analysed using open source Image J 1.44 software (NationalInstitute of Health, US).

Results

FIG. 4A shows that Fe-QA (“X”) at 100 μM inhibits the formation ofbiofilm by Pseudomonas aeruginosa. In the absence of Fe-QA, a highercoverage rate was measured for Pseudomonas aeruginosa than in thepresence of a 100 μM concentration of Fe-QA.

FIG. 4B shows that Fe-QA inhibits the formation of biofilm byUropathogenic E. coli (UPEC). In the absence of Fe-Q (“0 μM), a highercoverage rate is measured for UPEC than in the presence of 0.1 μM, 1 μM,10 μM and 100 μM concentrations of Fe-QA.

Example 5 FeQ prevents Attachment of Bacteria to Surfaces

Materials and Methods

Enteropathogenic E. coli (EPEC) E2348/69 were grown in wells for 48hours at 37° C. in the presence of FeQ (100 μM), and in the absence ofFeQ (as control). After 48 hours, the wells were washed in order toremove suspended cells. Crystal violet was then added to each well. Thewells were then washed to remove excess dye. A mixture ofacetone/ethanol was then added to the wells to re-suspend any cellsattached to the plastic surface of the wells, and dissolve any dyepresent. The presence of dye in each well was then quantified bymeasuring the O.D. at 570 nm.

Results

In the absence of FeQ, EPEC binds to the plastic surface and forms abiofilm that is readily detected by dying with crystal violet However,in the presence of FeQ, EPEC is unable to attach to the plastic surfaceand form a biofilm, and is not detected by adding crystal violet. FIG. 5shows quantitatively the difference in the attachment of EPEC cells tothe plastic well surface in the absence and presence of FeQ bymeasurement of the optical absorbance of crystal violet that wasabsorbed by EPEC cells attached to the surface. At an FeQ concentrationof 100 μM there is little or no attachment of bacterial cells to thesurface and no biofilm formation.

Example 6 FeQ prevents Formation of Biofilm on Human Teeth

Materials and Methods

Molar teeth were extracted from human patients, and mouth swabs takenfrom each patient to obtain samples of each patient's bacterial florapresent in the mouth. The mouth swabs were cultured in the laboratoryusing LB media in order to grow bacterial populations ordinarily presentin the mouth of each patient. The extracted teeth were washed andbrushed extensively using PBS buffer and ethanol. Each patient's toothwas then placed in the bacterial culture prepared from that patient'sbacterial flora sample, and cultured aerobically for 24 hours in LBmedia. The teeth were then stained with CEPLAC™ (Manx Healthcare Ltd,Warwick, UK), and washed three times with PBS-Tween (50 mL) to determineif biofilm had been formed on the teeth.

Results

All teeth stained red indicating the presence of biofilm on the teethafter just 24 hours. The same teeth were then cleaned using PBS bufferand ethanol, and the procedure repeated except with 48 hours ofculturing in the presence of FeQ (340 μM). After 48 hours, no teethstained red demonstrating that biofilms could not be established on theteeth in the presence of FeQ.

Example 7 Efficacy of FeQ and a Preparation Produced According toExample 14 (“FeTyr”) to Reduce Campylobater Carriage in Chickens andPromote Growth in Chickens

Materials and Methods

A study was performed to evaluate growth promotion and reduction ofCampylobater carriage using FeQ and FeTyr in Ross 308 male broilers with7 treatment groups. Each treatment group comprised four replicates of 10birds per pen (40 birds/treatment group and 4 pens of 10 birds/treatmentgroup), and there were 2 control groups and 5 test groups. All the testgroups and one of the control groups were exposed at day 20 of the trialto dirty litter, which tested positive for Campylobacter. This methodwas used to provide a more natural method to Campylobater challenge thebirds. Thus there was a positive control where one treatment group waschallenged with Campylobacter and one negative control group where thebirds were not challenged, and five treatment groups that were allchallenged with Campylobacter. The total number of birds used in the 7treatment groups was 280. Details of the treatments are provided inTable 1. Treatment group 1 was a negative control where birds justreceived the commercial feed, and were not challenged with dirty littercontaining Campylobacter. Treatment group 2 was the positive controlwhere the birds received the commercial feed, and were challenged withdirty litter containing Campylobater at day 20. Treatment group 3received 0.22 g/L of FeQ in their drinking water and 0.22 g/Kg FeQ intheir feed during the entire trial, and was challenged with dirty littercontaining Campylobater at day 20. Treatment group 5 received 0.22 g/Lof FeQ in their drinking water during the entire trial, and waschallenged with dirty litter containing Campylobater at day 20.Treatment group 6 received 0.22 g/kg FeQ in their feed during the entiretrial, and was challenged with dirty litter containing Campylobater atday 20. Treatment group 7 received 0.022 g/L FeQ in their drinking waterduring the entire trial, and was challenged with dirty litter containingCampylobater at day 20. Treatment group 8 received 0.02 g/L FeTyr intheir drinking water during the entire trial, and was challenged withdirty litter containing Campylobater at day 20. The FeTyr waspre-dissolved in DMSO, and diluted to provide a solution of 0.02 g/L ofFeTyr in water. (An additional treatment group 4 was terminated due tosolubility issues.)

TABLE 1 Treatment Details Campylobacter Treatment Description Challenge1 Control-1 Commercial feed No 2 Control-2 Commercial feed Yes 3 0.22g/L FeQ in water + Yes 0.22 g/kg FeQ in feed 5 0.22 g/L FeQ in water Yes6 0.22 g/kg FeQ in feed Yes 7 0.022 g/L FeQ in water Yes 8 0.02 g/LFeTyr in water Yes

The birds were fed with a commercial three-phase feeding program usingstarter, grower and finisher feeds with formulations shown in Table 2.All diets had coccidiostat (MAXIBAN® at 0.0625% in starter and finisherphase diets and MONTEBAN® at 0.06% in finisher phase). Xylanase(RONOZYME® WX at 200 g per ton) and phytase (RONOZYME® P at 150 gramsper ton) were added to all diets.

TABLE 2 Basal feed formulation for starter, grower and finisher dietsRaw Material STARTER % GROWER % FINISHER % Barley 10.5 8.4 7.2 Wheat50.0 55.0 60.0 Soya Ext Hipro 26.0 23.0 19.0 Full fat Soya Cherwell 5.05.0 5.0 L Lysine HCl 0.40 0.30 0.30 DL-methionine 0.40 0.35 0.30L-threonine 0.15 0.15 0.15 Soya Oil 4.0 4.50 4.75 Limestone 1.25 1.251.25 MonoCal phosphate 1.50 1.25 1.25 Salt 0.25 0.25 0.25 Sodiumbicarbarbonate 0.15 0.15 0.15 Broiler Premix 0.40 0.40 0.40 NutrientAnalysis Analysis Analysis Fat (ether extract) 6.34 6.85 7.11 Protein21.85 20.64 19.14 Fibre 3.08 3.02 2.97 Ash 6.01 5.68 5.50 ME-P 12.7813.04 13.22 Total lysine 1.45 1.28 1.17 Available lysine 1.35 1.19 1.09Methionine 0.69 0.62 0.55 Total methionine and 1.03 0.95 0.85 cysteineThreonine 0.91 0.86 0.79 Tryptophan 0.25 0.23 0.21 Calcium 0.95 0.910.89 Phosphorus 0.72 0.66 0.65 Available phosphorus 0.48 0.42 0.42 Salt0.30 0.30 0.30 Sodium 0.17 0.17 0.17 Vit A 13.20 13.5 13.50 Vit D3 5.05.0 5.00 Vit E 100 100 100

The feeding program is show in Table 3. The birds were reared in floorpens to day 42, and fed starter, grower and finisher feed at day 0 to11, 11 to 24, and 24 to 42 days, respectively. All birds were weighedindividually and feed weigh backs recorded per pen at day 0, 11, 21, 24and 42 days.

TABLE 3 Feeding Program Feeding Phase Starter Grower Finisher (days ofage) 0-11 11-24 24-42

Prior to challenging the chickens with dirty litter containingCampylobacter at day 20, each pen was tested for Campylobater usingcloacal swabs. All pens tested negative for Campylobater prior to thechallenge. At day 20, litter, which was naturallyCampylobacter-contaminated was tested to confirm the presence ofCampylobacter, and then added (approximately 2 kg/pen) to the litter inall pens except in pens for treatment group 1 (the negative control). Atday 28, the pen litter was sampled to confirm the presence or absence ofCampylobacter. At day 41 and 42, caecal samples were taken from 3 birdsper pen (12 birds per treatment group) and tested for Campylobaterenumeration. At day 42, digesta, fecal samples, and caecal content wastaken from all birds, and pooled per pen. Two birds per pen were alsotaken from treatment groups 1-3, euthanized, and blood samples taken.Samples were analyzed for blood chemistry, including analysis foralkaline phosphatase, aspartate amino transferase, alanine aminotransferase, gamma-glutamyl transferase, lactate dehydrogenase, totalprotein, albumin, globulin, amylase and glucose.

In order to minimize risk of cross-contamination, standard industrybiosecurity measures were used including: disinfecting boots, changingovershoes and gloves between pens/treatments, entering Campylobacternegative pens before entering Campylobater positive pens, and leavingadjacent pens empty. Daily health, culls, and mortality were recorded.All bird weights were recorded at 0, 11, 21, 24, 33 and 42 days. Weightgains, feed intake and feed conversion ratio (FCR) were derived for eachfeeding period.

The effect of the treatment groups compared to the negative controlgroup (treatment group 1) and the positive control group (treatmentgroup 2) is shown in Tables 5-12 for the periods 0-11 days, 11-20 days,20-25 days, 11-25 days, 25-42 days, 20-42 days, 0-20 days, and 0-42days.

Results

FIG. 8 shows the average body weight at day 42 for all treatment groups,and a comparison to a commercial control labeled “Target”. The figureshows that treatment group 1 (the negative control labeled “CNC”)attained an average body weight (ABW) of 3.437 kg at day 42 (which washigher than the commercial target of 2.979 kg). The positive control(labeled “CC”), which was challenged with dirty litter containingCampylobater at day 20, in contrast only attained an ABW of 3.186 kg atday 42, which was significantly less than the negative control(treatment group 1). This result demonstrates that challenging withdirty litter contaminated with Campylobacter resulted in a reduction ofgrowth of the chicken by an average of 251 grams. However, when thechickens were challenged with dirty litter containing Campylobater buttreated with FeQ or FeTyr in treatment groups 3, 5, 6, 7 and 8, alltreatment groups performed better than the positive controldemonstrating that FeQ and FeTyr treatment had a positive effect ongrowth. In fact, FeQ in feed at 0.22 g/kg (treatment group 6) producedchicken with an ABW of 3.464 kg, which was higher than the negativecontrol ABW of 3.437 kg even though treatment group 6 had beenchallenged with dirty litter containing Campylobacter.

FIG. 9 shows the mortality adjusted feed conversion rate (MFCR) at day42 for all treatment groups, and a comparison to a commercial controllabeled “Target”. (A lower MFCR number is a better result.) The figureshows that treatment group 1 (the negative control labeled “CNC”) had aMFCR of 1.563, which was lower than the commercial target of 1.703. Thepositive control, labeled “CC” which was challenged with the dirtylitter containing Campylobater at day 20 had a significantly higher MFCRof 1.679 than the negative control. Thus challenging with dirty litterinfected with Campylobater resulted in a higher MFCR. However, when thechickens were challenged with dirty litter infected with Campylobaterbut treated with FeQ or FeTyr in treatment groups 3, 5, 6, 7 and 8, alltreatment groups performed better than the positive controldemonstrating that FeQ and FeTyr treatment had a positive effect on MFCR(i.e. decreasing the numerical MFCR). The results show that treatmentgroups 3, 5, 6, 7 and 8 had MFCR values of 1.595, 1.560, 1,563, 1.612and 1.577, respectively. Furthermore, treatment groups 5 and 6 performedas well as the negative control even when challenged with dirty littercontaining Campylobacter.

FIG. 10 shows the number of Campylobater colony forming units per gram(cfu/g) of bird droppings at day 42 for treatment groups 1-3 and 6-8. (Alower number is a better result.) The results show that treatment groups3 and 6-8 all performed better than the positive control (treatmentgroup 2) demonstrating that FeQ and FeTyr had a positive effect onreducing Campylobacter infection of poultry. Notably, chicken treatedwith FeTyr, FeQ in feed, and FeQ in feed and water all had colonyforming units of Campylobacter per gram of dropping that were similarto, or less than, those of the negative control group (treatment group1). The detection of low levels of Campylobater in the negative controlsdemonstrates how highly contagious the bacterium is, and is likely to bean indication that a small number of birds in the negative control groupbecame infected despite not being experimentally challenged with dirtylitter. The results in FIG. 10 for the low concentration of FeQ in water(0.022 g/L; treatment group 7) appears to show less of an effect thanthe other treatment groups, although this difference was considered morelikely due to experimental error for example following crosscontamination of samples. As discussed below, the results obtained froma further experiment, as given in FIG. 11 confirm that treatment group 7did, indeed, also provide the highly beneficial effect.

FIG. 11 shows the average number of Campylobater colony forming unitsper gram (cfu/g) of caeca samples at day 42 for treatment groups 1-3 and5-8. The results show that all the treatment groups (3 and 5-8) allperformed better than the positive control (treatment group 2)demonstrating that FeQ and FeTyr had a positive effect on reducingCampylobacter infection of poultry.

The effect of the treatments on overall liveability and Europeanproduction and efficiency factor (EPEF) is shown in Table 4. (EPEF=[(Liveability×Live weight in kg at end of trial/Age in Days×FCRcommercial)×100].

The effect of FeQ treatment on growth performance in the absence ofCampylobacter challenge during the starter phase (0-11 days) and periodfrom 0-20 days is shown in Table 13. Since the negative and positivecontrols (treatment groups 1 and 2) are identical prior to challengewith the dirty litter at day 20, these groups may be pooled forcomparison to treatment groups 3, 5, 6, and 7 in order to see if FeQ hadan effect on growth in the absence of a challenge by dirty littercontaminated with Campylobacter l during the first 20 days of growth.The results demonstrate that FeQ promotes growth of chicken even in theabsence of a challenge from dirty litter contaminated withCampylobacter. At day 20, the average body weight (ABW) for the controlgroups (treatment groups 1 and 2) is 0.927 kg versus 0.963 kg fortreatment groups 3, 5, 6 and 7 which all received FeQ. This improvementin body weight is also reflected in a significantly better MFCR for theFeQ treated birds. Table 10 shows the MFCR for the birds treated ingroups 3, 5, 6 and 7 is 1.2996 versus 1.3374 for the control groups(treatment groups 1 and 2). Notably the P-value is less than 0.05.

The same positive effect of FeTyr treatment on growth performance in theabsence of Campylobater challenge is also evident from Table 10. The AWGduring the first 20 days of production for chicken treated with FeTyr(treatment group 8) is 0.895 kg compared to 0.884 and 0.889 kg fortreatment groups 1 and 2 (negative and positive controls). Furthermore,the MFCR during the first 20 days of production for chicken treated withFeTyr (treatment group 8) is 1.311 versus 1.32 and 1.355 for treatmentgroups 1 and 2, respectively. (A lower MFCR value is an improvement.)

The results of this study demonstrate that both FeQ and a preparationproduced according to the method of Example 14 (FeTyr) promote growthand decrease the mortality adjusted feed conversion ratio (MFCR) in theabsence or presence of dirty litter contaminated with Campylobacter.

TABLE 4 Effect of treatments on growth performance during starter phase(day 0-11) ABW AFD AWG MFCR Treatment Day 0 Day 11 Day 0-11 1 0.0400.331 0.348 0.291 1.239^(b) 2 0.040 0.337 0.359 0.297 1.228^(b) 3 0.0400.346 0.356 0.306 1.181^(ab) 5 0.040 0.334 0.352 0.294 1.210^(ab) 60.041 0.351 0.360 0.310 1.168^(a) 7 0.040 0.325 0.348 0.285 1.236^(b) 80.040 0.329 0.353 0.289 1.229^(b) P-value 0.136 0.418 0.979 0.463 0.005SED 0.000 0.013 0.016 0.013 0.018 P-value for contrast 1 vs 2 0.5120.667 0.519 0.682 0.584 1 vs 2 to 8 0.666 0.573 0.603 0.583 0.045 2 vs3567 0.632 0.844 0.723 0.834 0.054 5 vs 6 0.099 0.213 0.627 0.233 0.0335 vs 7 0.141 0.466 0.804 0.494 0.170 2 vs 8 0.645 0.538 0.709 0.5490.982 ^(a-b)within a column reflects differences between treatments whenP < 0.05; SED = Standard errors of difference of means; ABW = averagebody weight (kg); AFD = average feed intake (kg); AWG = average weightgain (kg); MFCR = Mortality adjusted feed conversion ratio; FCR = Feedconversion ratio-commercial.

TABLE 5 Effect of treatments on growth performance during grower phase(day 11-20) ABW AFD AWG MFCR Treatment Day 20 Day 11-20 1 0.924 0.7990.593 1.362 2 0.929 0.838 0.592 1.421 3 0.972 0.857 0.625 1.375 5 0.9430.821 0.609 1.348 6 0.991 0.841 0.640 1.343 7 0.947 0.829 0.622 1.333 80.935 0.809 0.606 1.351 P-value 0.358 0.311 0.279 0.279 SED 0.032 0.0250.021 0.036 P-value for contrast 1 vs 2 0.875 0.133 0.977 0.115 1 vs 2to 8 0.248 0.094 0.175 0.987 2 vs 3567 0.189 0.961 0.075 0.020 5 vs 60.145 0.427 0.160 0.884 5 vs 7 0.913 0.737 0.546 0.673 2 vs 8 0.8500.253 0.516 0.065 SED = Standard errors of difference of means; ABW =average body weight (kg); AFD = average feed intake (kg); AWG = averageweight gain (kg); MFCR = Mortality adjusted FCR; FCR = FCR commercial.

TABLE 6 Effect of treatments on growth performance during period day20-25. ABW AFD AWG MFCR Treatment Day 25 Day 20-25 1 1.366 0.662 0.4421.500 2 1.371 0.652 0.442 1.550 3 1.424 0.667 0.453 1.477 5 1.384 0.6580.441 1.495 6 1.426 0.685 0.434 1.599 7 1.388 0.661 0.441 1.513 8 1.3770.662 0.442 1.499 P-value 0.723 0.916 0.999 0.882 SED 0.044 0.026 0.0300.096 P-value for contrast 1 vs 2 0.912 0.685 0.998 0.604 1 vs 2 to 80.403 0.932 0.996 0.759 2 vs 3567 0.339 0.444 0.990 0.707 5 vs 6 0.3610.311 0.826 0.294 5 vs 7 0.930 0.902 0.988 0.854 2 vs 8 0.892 0.6930.999 0.604 SED = Standard errors of difference of means; ABW = averagebody weight (kg); AFD = average feed intake (kg); AWG = average weightgain (kg); MFCR = Mortality adjusted FCR; FCR = FCR commercial.

TABLE 7 Effect of treatments on overall growth performance during growerphase (day 11-25) AFD AWG MFCR Treatment Day 11-25 1 1.462 1.035 1.421 21.490 1.034 1.457 3 1.524 1.078 1.417 5 1.479 1.050 1.409 6 1.526 1.0751.440 7 1.490 1.064 1.406 8 1.471 1.048 1.414 P-value 0.660 0.804 0.598SED 0.042 0.036 0.030 P-value for contrast 1 vs 2 0.516 0.984 0.241 1 vs2 to 8 0.293 0.406 0.891 2 vs 3567 0.657 0.267 0.118 5 vs 6 0.280 0.4980.300 5 vs 7 0.787 0.707 0.925 2 vs 8 0.664 0.695 0.165 SED = Standarderrors of difference of means; ABW = average body weight (kg); AFD =average feed intake (kg); AWG = average weight gain (kg); MFCR =Mortality adjusted FCR.

TABLE 8 Effect of treatments on overall growth performance duringfinisher phase (day 25-42) ABW AFD AWG MFCR Treatment Day 42 Day 25-42 13.437 3.479 2.070^(b) 1.688 2 3.186 3.480 1.814^(a) 1.889 3 3.342 3.3871.918^(ab) 1.773 5 3.407 3.357 2.023^(b) 1.706 6 3.464 3.315 2.039^(b)1.704 7 3.304 3.362 1.916^(ab) 1.793 8 3.341 3.434 1.964^(ab) 1.716P-value 0.027 0.56 0.009 0.211 SED 0.075 0.099 0.062 0.081 P-value forcontrast 1 vs 2 0.004 0.997 <.001 0.022 1 vs 2 to 8 0.110 0.247 0.0160.233 2 vs 3567 0.004 0.129 0.004 0.035 5 vs 6 0.455 0.680 0.800 0.988 5vs 7 0.187 0.960 0.101 0.294 2 vs 8 0.053 0.649 0.027 0.046 ^(a-b)withina column reflects differences between treatments when P < 0.05; SED =Standard errors of difference of means; ABW = average body weight (kg);AFD = average feed intake (kg); AWG = average weight gain (kg); MFCR =Mortality adjusted FCR; FCR = FCR commercial.

TABLE 9 Effect of treatments on the growth performance during theexperimental period of day 20-42 (after the birds were challenged) AFDAWG MFCR Treatment Day 20-42 1 4.142 2.512^(b) 1.653 2 4.131 2.256^(a)1.820 3 4.054 2.370^(ab) 1.713 5 4.015 2.464^(ab) 1.665 6 4.0012.473^(ab) 1.678 7 4.023 2.357^(ab) 1.739 8 4.096 2.406^(ab) 1.676P-value 0.767 0.025 0.344 SED 0.110 0.068 0.075 P-value for contrast 1vs 2 0.926 0.001 0.038 1 vs 2 to 8 0.306 0.028 0.290 2 vs 3567 0.2290.008 0.055 5 vs 6 0.898 0.894 0.856 5 vs 7 0.941 0.138 0.331 2 vs 80.752 0.042 0.070 ^(a-b)within a column reflects differences betweentreatments when P < 0.05; SED = Standard errors of difference of means;ABW = average body weight (kg); AFD = average feed intake (kg); AWG =average weight gain (kg); MFCR = Mortality adjusted.

TABLE 10 Overall effect of treatments on growth performance during theexperimental period of day 0-20 (before birds were challenged). AFD AWGMFCR Treatment Day 0-20 1 1.147 0.884 1.320 2 1.196 0.889 1.355 3 1.2130.931 1.310 5 1.173 0.903 1.303 6 1.201 0.951 1.284 7 1.178 0.907 1.3028 1.161 0.895 1.311 P-value 0.623 0.368 0.225 SED 0.038 0.032 0.025P-value for contrast 1 vs 2 0.215 0.881 0.181 1 vs 2 to 8 0.191 0.2510.627 2 vs 3567 0.860 0.188 0.012 5 vs 6 0.476 0.150 0.469 5 vs 7 0.9070.899 0.978 2 vs 8 0.371 0.846 0.094 SED = Standard errors of differenceof means; ABW = average body weight (kg); AFD = average feed intake(kg); AWG = average weight gain (kg); MFCR = Mortality adjusted.

TABLE 11 Overall effect of treatment groups on growth performance (day0-42) AFD AWG MFCR Treatment Day 0-42 1 5.289 3.397^(b) 1.563 2 5.3283.145^(a) 1.679 3 5.267 3.302^(ab) 1.595 5 5.188 3.367^(ab) 1.560 65.201 3.423^(b) 1.563 7 5.201 3.265^(ab) 1.612 8 5.258 3.301^(ab) 1.577P-value 0.920 0.028 0.193 SED 0.132 0.075 0.047 P-value for contrast 1vs 2 0.773 0.004 0.024 1 vs 2 to 8 0.633 0.111 0.352 2 vs 3567 0.290.004 0.018 5 vs 6 0.920 0.461 0.954 5 vs 7 0.924 0.190 0.284 2 vs 80.601 0.053 0.043 ^(a-b)within a column reflects differences betweentreatments when P < 0.05; SED = Standard errors of difference of means;ABW = average body weight (kg); AFD = average feed intake (kg); AWG =average weight gain (kg); MFCR = Mortality adjusted.

TABLE 12 The effect of treatments on overall liveability and Europeanproduction and efficiency factor (EPEF) EPEF Treatment Day 20 Day 42 1318.3 282.8 2 334.7 250.7 3 350.4 262.9 5 352.0 278.3 6 364.8 265.0 7354.5 276.2 8 336.4 296.0 P-value 0.547 0.842 SED 23.83 31.68 P-valuefor contrast 1 vs 2 0.500 0.323 1 vs 2 to 8 0.111 0.645 2 vs 3567 0.2850.437 5 vs 6 0.599 0.680 5 vs 7 0.919 0.949 2 vs 8 0.945 0.170

TABLE 13 Effect of treatments on growth performance in absence ofCampylobacter challenge during starter phase (0-11 days) and period 0-20days. ABW ABW AFD AWG MFCR Treatment Day 11 Day 20 0-20 days 0-20 days0-20 Groups 1 & 2 0.334 0.927 1.172 0.887 1.3374 FeQ 0.339 0.963 1.1910.923 1.2996 (Groups 3, 5, 6, 7) P-value 0.584 0.079 0.432 0.078 0.029SED 0.009 0.020 0.024 0.020 0.016 ABW = average body weight (kg); AFD =average feed intake (kg); AWG = average weight gain (kg); MFCR =mortality adjusted feed conversion ratio

Example 8 FeDOPA Prevents Attachment of Bacteria to Surfaces

Materials and Methods

Enteropathogenic E. coli (EPEC) E2348/69 were grown in wells for 48hours at 37° C. in the presence of FeDOPA (10-250 μM), and in theabsence of FeDOPA (as control). After 48 hours, the wells were washed inorder to remove suspended cells. Crystal violet was then added to eachwell. The wells were then washed to remove excess dye. A mixture ofacetone/ethanol was then added to the wells to re-suspend any cellsattached to the plastic surface of the wells, and dissolve any dyepresent. The presence of dye in each well was then quantified bymeasuring the O.D. at 570 nm.

Results

In the absence of FeDOPA, EPEC binds to the plastic surface and forms abiofilm that is readily detected by dying with crystal violet. However,in the presence of FeDOPA, EPEC attachment to the plastic surface andformation of a biofilm is inhibited. FIG. 12 shows quantitatively thedifference in the attachment of EPEC cells to the plastic well surfacein the absence and presence of FeDOPA by measurement of the opticalabsorbance of crystal violet that was absorbed by EPEC cells attached tothe surface. At an FeDOPA concentration of 68-250 μM attachment ofbacterial cells to the surface and biofilm formation is inhibited.

Example 9 Disruption of a Preformed Biofilm with a Preparation ProducedAccording to the Method of Example 14 (“FeTyr”)

Crystal violet assays were used as described above to demonstrate thatFeTyr could disrupt a pre-formed biofilm. A mature biofilm formed byEPEC-pgA⁺⁺ was treated with FeTyr for 24, 48 and 72 hours at FeTyrconcentrations of 100 μM, 150 μM and 200 μM and the presence of thebiofilm after these times was compared to an untreated biofilm (labeled“Control”) using a crystal violet assay. The color of the control wellswas more intense in color at 72 hours than those that were treated withFeTyr at 100, 150 and 200 μM for 72 hours. FIG. 13 shows quantitativelythe optical absorbance of crystal violet at 570 nm that was absorbed bythe EPEC cells that remained attached to the surface of the plastic wellafter a mature biofilm formed by EPEC-pgA⁺⁺ was treated with FeTyr(shown as FeY in FIG. 13) at 100 μM, 150 μM and 200 μM compared to anuntreated biofilm (labeled “Control”) in the crystal violet assay. Asignificantly lower optical absorbance was found at 72 hours for thebiofilm treated with FeTyr at 100, 150 and 200 μM at 72 hours. Theseresults demonstrate that a preparation produced according to the methodof Example 14 (“FeTyr”) can disrupt a pre-formed biofilm.

Example 10 Disruption of a Preformed Biofilm with a Preparation ProducedAccording to the Method of Example 14 (“FeTyr”) and Fe-DOPA

A BioFlux system was used to demonstrate that a preparation producedaccording to the method of Example 14 (“FeTyr”) and Fe-DOPA can be usedto disrupt a mature EPEC-ΔcsrA biofilm. Our studies showed that that amature biofilm of EPEC-ΔcsrA can be formed in the presence of LB medium30% v/v and imaged (data not shown). The mature biofilm was treated withFeTyr at concentrations of 100, 150 and 200 μM for 20 hours and comparedto a control biofilm that had just been treated with LB medium 30% v/v.It was found that biofilm dispersion increased as the concentration ofFeTyr was increased from 100 to 200 μM (data not shown). Mature biofilmwas treated with FeDOPA at a concentration of 100 μM for 20 hours andcompared to a control biofilm that had just been treated with LB medium30% v/v. It was found that Fe-DOPA dispersed the biofilm at aconcentration of 100 μM (data not shown).

Example 11 Treatment of an Acne Patient with FeQ

An 18 year old female patient was treated continuously for 30 days byapplying a solution of ferric quinate (340 μM) to her acne vulgarism(“acne”) once each day. Within 5 days of the start of treatment, heracne, which had not previously responded to treatment with antibiotics,began to show signs of healing. After treatment for 30 days, her acnewas completely healed. Her acne did not recur even after discontinuationof treatment for over one year.

Example 12 Effect of FeQ on Biofilm Formation of a Medical Device

Materials and Methods

To investigate the effect of Ferric Quinate (FeQ) on the surfaceintegrity of contact lenses, two contact lenses were independentlyincubated in either saline solution, or saline solution with a finalconcentration of 340 μM FeQ at 4° C. for 7 days, whilst gently shaking.The lenses were then washed 6 times with phosphate-buffered solution(PBS)+0.05% Tween. Each lens was then washed twice with distilled waterbefore analysis via environmental scanning electron microscope (ESEM).

To investigate biofilm formation on the contact lens, clinicallydetermined PAO-1 strains of Pseudomonas aeruginosa were incubated withthe lenses in either Luria-Bertani Media (LB) or LB with a finalconcentration of 340 μM FeQ at 37° C. for 24 hours in a non-shakingincubator. The lenses were then washed 6 times with PBS+0.05% Tween,before being stored overnight in PBS+0.05% Tween. In preparation for theESEM, formaldehyde was added to a final concentration of 1% andincubated for 10 minutes in order to inactivate the bacteria. The lenseswere then washed 4 times with PBS+0.05% Tween, and immediately beforeanalysis the lenses were washed a further two times with distilledwater.

Results

Surface Integrity

Lenses were treated with 340 μM FeQ or left untreated (control) toinvestigate the effect, if any, of FeQ treatment on the surfaceintegrity of the contact lens.

The results show that FeQ at 340 μM does not have any visiblesignificant effect (via ESEM) on the surface integrity of the contactlens.

Biofilm Formation

ESEM images (data not shown) following incubation with bacteria onlyshowed large scale biofilm development of Pseudomonas aeruginosa, overthe surface of the contact lens. Analysis via the ESEM requires vacuumand causes areas of the biofilm to dehydrate, which is responsible forthe perforated appearance of the biofilm of the contact lens. Thedehydration gives perception of depth, and shows the biofilm formed inthe absence of FeQ to be substantial. Individual bacteria were visiblein the biofilm, surrounded by the extracellular matrix (ECM).

The impact of incubation with bacteria in the presence of FeQ 340 μM wasalso investigated. These images (data not shown) showed that, in thepresence of FeQ at 340 μM, Pseudomonas aeruginosa appears as eithersingle bacterium, or small aggregates of bacteria, with no apparent ECMformation. These results indicate that FeQ inhibits biofilm formation ofPseudomonas aeruginosa.

Based on these results, it can be concluded the FeQ and other compoundsand preparations as described herein can be used to inhibit or preventbiofilm formation on medical devices, such as but not limited to,contact lenses.

Example 13 Preparation Protocol for K [Fe (C₇H₁₁O₆)₃] (OH) 3H₂O (FeQ)

FeCl₃.6H₂O (50 g, 184 mmol, Alfa Aesar, 97%) was placed in a flask anddissolved in 300 mL of H₂O (J. T. baker, HPLC grade). To that solution,D-(−)-quinic acid (110 g, 572 rnmol, Buchlr Gmbh, 96%) was added slowlywith continuous stirring. The pH of the solution was adjusted to ˜3 byaddition of 10M KOH (Alfa Aesar, 85%) (˜80 mL was required).

The dark yellow solution darkened to brownish upon addition of KOH. Thedark solution was stirred for 1 h at room temperature. After stifling atroom temperature for 1 h ethanol (EMD, 94%) (2.5 l ) was added slowly tothe solution with stirring.

After addition of approximately ¼ of the total ethanol, the solutionlightened visibly and a fine solid began to precipitate from solution.After addition of the remaining ethanol, the solution is allowed to sitovernight at room temperature.

The solids are collected by vacuum filtration on a fritted funnel andallowed to dry on the funnel while the vacuum is continued for 2-3 h.The bright yellow solid is spread in a thin layer in a drying dish anddried open to the air for 3 days followed by drying under vacuum for 48h to give 155 g of the final product.

Example 14 Exemplary Method for the Synthesis of a Preparation Accordingto the Present Invention (“FeTyr”)

A preparation was prepared by a method in which L-tyrosine and LiOH.H₂Owere dissolved in water and the solution heated. FeCl₃ salt wasdissolved in water and was added to the tyrosine/LiOH solution.Precipitation (brown solid) was almost instantaneous but stirring withheating continued. The product was then allowed to cool and wascollected by filtration. The product was air dried and then furtherdried, for example using a lyophilizer.

More specifically, in one particular example of the aforementionedmethod, L-tyrosine (5.43 g, 30 mmol, Chem Impex, 99.5%) and LiOH.H₂O(1.26 g, 30 mmol, EMD, 94%) were dissolved in water (250 ml, J. T.Baker, HPLC grade)) and the solution heated to 70° C. for 20 min. TheFeCl₃ salt (1.62 g, 10 mmol, Alfa Aesar, 98%)) was dissolved in aminimum quantity of water (3-5 ml) and was added to the tyrosine/LiOHsolution.

Precipitation (brown solid) was almost instantaneous but stirring withheating continued, such as for 15 min. The product was allowed to coolto room temperature and was collected by filtration. The product was airdried and then further dried in a lyophilizer Isolated yield obtainedfrom the specific method as described in the foregoing paragraph was5.85 g.

Preliminary elemental analysis of the product produced by the specificmethodology described above showed: carbon 49.19%, hydrogen 5.37%,nitrogen 6.33%, oxygen 30.48% and iron 8.63%.

Further preliminary analysis of the product, as produced by the specificmethodology described above, indicated:

-   -   Ignition on loss (ash) 550° C. for 6 hours: 13.32%    -   Moisture, vacuum, 100° C., 5 hours 2.23%

Example 15 MALDI-TOF Analysis of an Exemplary Preparation According tothe Present Invention

The product, as produced by the specific methodology described inExample 14 was further assessed using MALDI-TOF mass spectrum. Anobjective of the analysis was to confirm the molecular weight of a 1:3complex of iron (III) and tyrosine.

Summary and Conclusions

The MALDI-TOF mass spectrum of the sample contained a peak at 619.37Da., consistent with the expected iron (III) and tyrosine complex (in aratio of 1:3) plus sodium cationization.

A number of other unidentified peaks were also seen in the 400 to 700 Damass range. Two of the unidentified peaks may be related to the 1:3 ironto tyrosine complex or other complex, hydrate or salt formation betweeniron and tyrosine. These appear at around 591 and around 647 Da., adifference of 28 Da. less and more than the complex, respectively. Thestructures corresponding to these peaks are not known, and may representunidentified structures within the preparation.

Example 16 Alternative Exemplary Method for the Synthesis of aPreparation According to the Present Invention (“FeTyr”)

L-tyrosine and lithium hydroxide monohydrate were dissolved in water andthe solution heated. Anhydrous iron (III) chloride was dissolved inwater and was added to the tyrosine/LiOH solution. Precipitation (brownsolid) was almost instantaneous but stirring with heating continued. Theproduct was allowed to cool to room temperature and collected byfiltration. The product was then washed with warm water, air dried, andthen further dried, for example using a vacuum oven.

More specifically, in one particular example of the aforementionedmethod, L-tyrosine (330 g, 1.82 mol, Chem Impex, Catalogue #00304,99.5%) and lithium hydroxide monohydrate (76.5 g, 1.82 mol, EMD,Catalogue #LX0350-5, 94%) were dissolved in water (17 L) and thesolution heated to 70° C. for 20 min Anhydrous iron (III) chloride (97.5g, 0.6 mol, Alfar Aesar, Product #12357, 98%) was dissolved in 100 ml ofwater and was added to the tyrosine/LiOH solution. Precipitation (brownsolid) was almost instantaneous but stirring with heating continued for15 min. The product was allowed to cool to room temperature overnightand was collected by filtration. The product was washed with warm water(70° C., 2 L), air dried for 2 days and then dried in a vacuum oven at40° C. for 2 days. Typical isolated yield was 350 g (98% yield).

Example 17 Further Alternative Exemplary Method for the Synthesis of aPreparation According to the Present Invention (“FeTyr”)

FeCl₃ was added to a suspension of tyrosine and Et₃N in methanol andrefluxed. The colour changed to violet immediately upon addition. Thesolid was filtrated and air-dried and then further dried, for example,over CaCl₂ under vacuum.

More specifically, in one particular example of the aforementionedmethod, FeCl₃.6H₂O (1 mmol, 0.27 g) was added to a suspension oftyrosine (3 mmol, 0.543 g) and Et₃N (3 mmol, 0.303 g, 0.41 mL) in 100 mLmethanol at 75° C. and refluxed for 24 hrs. The colour changed to violetimmediately upon addition. The solid was filtered and air-dried and thendried over CaCl₂ under vacuum.

Example 18 Synthesis of Fe(DOPA)₃

L-Dopa (11.84 g, 60 mmol, AK Scientific, 98%) and LiOH.H₂O (2.52 g, 60mmol, EMD, 94%) were dissolved in water (100 ml, J. T. Baker, HPLCgrade) and the solution heated to 70° C. for 20 mm The FeCl₃ salt (3.2g, 20 mmol, Alfa Aesar, 98%) was dissolved in a minimum quantity ofwater (6-10 ml) and was added to the Dopa/LiOH solution vigorous.

Precipitation (very dark purple) was almost instantaneous but stirringwith heating continued for 15 min.

The product was allowed to cool to room temperature and was collected byfiltration. The product was air dried and then further dried in alyophilizer Isolated yield was 6.5 g. More solid (4 g) was collectedfrom the filtrate and dried in the same way. Overall yield was 10.5 g.

Example 19 Fe-Q and Fe-Phe Potentiate the Effect of Antibiotics

Methods

To investigate effects upon antibiotic resistance, a laboratory strainof Pseudomonas aeruginosa (PAO1N) and a mixed population of clinicalisolates (PAO Mixed) were incubated in Luria-Bertani (LB) media alone,or with different concentrations (34 μM, 100 μM, 200 μM and 340 μM) ofFeQ or FePhe.

Each of the different media, bar one control, contained 10 μg/ml of theaminoglycoside antibiotic Amikacin.

10 μl of the bacterial strains were added into each well of a 96-wellmicro-titer plate, before 290 μl of the relevant media was added towells. Each different condition was repeated in sextuplicate.

The plate was incubated at 37° C. within a micro-titer plate reader for17.5 hours, with the OD₆₀₀ read every 30 minutes.

Results

The results are shown in FIGS. 14A and 14B. These figures show that Fe-Qand Fe-Phe provide similar effects at reducing tolerance of PAO1N andPAO Mixed to the aminoglycoside Amikacin

Example 20 Further Alternative Exemplary Method for the Synthesis of aPreparation According to the Present Invention (“FeTyr”)

Methods

L-tyrosine (50 g, 0.275 mol) and lithium hydroxide monohydrate (11.57 g,0.275 mol) were suspended in water (2.6 L, 52 Vol.) and the mixture washeated to 70 -75° C. for 30 min. The solution became, clear andhomogeneous.

Iron (III) chloride hexahydrate (24.6 g, 0.091 mol) was dissolved inwater (15 mL, 0.3, Vol.) at ambient temperature. The solution wasfurther diluted with water (85 Ml, 1.7 Vol.) to enable gradual andaddition of aqueous iron (III) chloride solution into the pre-dissolvedtyrosine/LiOH aqueous solution at 70 -75° C. over a period of time ofless than 5 minutes. Aqueous FeCl₃ solution was added into tyrosinesolution at 70 -75° C. over a period of time of less than 5 minutes.During the addition of aq. FeCl₃ solution, precipitation was observed.

The heating mantle was turned off and the reaction mixture was graduallyallowed to attain room temperature. An increase in the precipitation wasobserved as the reaction mixture was cooled. The suspension was stirredfor 16 -18 h at 25 -35° C. after which the precipitation of the productwas completed.

Afterwards, the filtration of the suspension and washing of the wet cakewith warm water (˜70° C.; 300 mL, 6 Vol.) and air drying of the isolatedmaterial for ˜16 h followed by vacuum drying at below 45° C. for 2 -4 hwas performed to obtain 49 g of the Fe-Tyr product.

Results

Characterization:

Elemental Analysis:

Elemental analysis (see Table 14 below) suggested that FeTyr produced bythe instant method was not simply made up of an Fe(tyr)₃ compound but ofa mixture which included an Fe(tyr)₃ compound. Based on the low carbonand high oxygen in the “found” analysis numbers, water was added to theformula. Addition of three waters of hydration showed a better carbonand hydrogen match with oxygen being low. Iron carboxylates often formtrimers, with a triply-bridging oxygen at the center. Keeping the samerelative Fe/tyrosine/water ratio, but formulating it as a μ-oxo trimer,the elemental analysis is within acceptable limits for all elements.

TABLE 14 Elemental Analysis Data for FeTyr product H C N O Fe Found 5.3749.19 6.33 30.48 8.63 Fe(tyr)₃ 5.07 54.38 7.05 24.14 9.36 Fe(tyr)₃•3H₂O5.58 49.86 6.46 29.52 8.59 Fe₃O(tyr)₉•9H₂O 5.53 49.45 6.41 30.09 8.52

We claim:
 1. A method of making an Fe(III) complex, the methodcomprising the steps of: bringing a tyrosinate moiety into contact withan Fe(III)-containing substance in the presence of a base.
 2. The methodof claim 1, wherein the tyrosinate moiety is tyrosine or a tyrosinesalt.
 3. The method of claim 1, wherein the Fe(III)-containing substanceis selected from the group consisting of FeCl₃, FeBr₃, Fe(OH)₃,Fe₂(SO₄)₃, FePO₄, Fe(HSO₄)₃, Fe(NO₃)₃, Fe(acetylacetonate)₃(“Fe(acac)₃”), Fe(acetate)₃ and Fe(lactate)₃, and salts and hydratesthereof.
 4. The method of claim 1, wherein the Fe(III)-containingsubstance is iron (III) chloride hexahydrate.
 5. The method of claim 1,wherein the bringing step is occurs in less than about 5 minutes.
 6. Themethod of claim 1, wherein the base is an amine, a nitrogen-basedheterocycle, an alkali metal alkoxide, an ammonium or alkali metalhydroxide, or an alkaline earth or an alkali metal carbonate orbicarbonate.
 7. The method of claim 1, wherein the tyrosine or othertyrosinate moiety is predominantly L-tyrosine or an L-tyrosinate moiety.8. The method of claim 1, wherein the method does not include a step ofisolating Fe(tyrosine)₃ from one or more components of the reactionmixture.
 9. The method of claim 1, wherein the preparation comprisesFe(OH)₃.
 10. A product selected from the group consisting of apharmaceutical,veterinary product, medical device, and dietary product,wherein the product comprises the Fe(III) complex prepared by the methodof claim
 1. 11. A product selected from the group consisting of apharmaceutical or veterinary product, a medical device or a dietaryproduct comprising a combination of the Fe(III) complex preparedaccording to claim 1 and one or more antimicrobial agents.
 12. Theproduct of claim 11, wherein the amount or concentration of the one ormore antimicrobial agents in the product is 5%, 10%, 15%, 20%, 30%, 40%,50%, 60%, 70%, 80% or more, less than a therapeutically effective ortherapeutically optimal amount or concentration of the one or moreantimicrobial agents when administered to the patient or animal that isnot in receipt of the product.
 13. The product of claim 11, wherein theone or more antimicrobial agents are antibiotics, for exampleantibiotics selected from the group consisting of aminoglycosides,ansaycins, carbapenems, cephalosporins, glycopeptides, lincosamides,lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones,penicillins, polypeptides, quinolones/fluoroquinolone, sulfonamides,tetracyclines, clofazimine, dapsone, capreomycin, cycloserine,ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin (rifampin),rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol,fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin,quinupristin/dalfopristin, thiamphenicol, tigecycline, tinidazole, andtrimethoprim; and combinations thereof.
 14. A composition comprising anFe(III) complex prepared by a method comprising the steps of: bringing atyrosinate moiety into contact with an Fe(III)-containing substance inthe presence of a base; optionally wherein the composition is incombination with one or more antimicrobial agents.
 15. The compositionof claim 14 for treatment or prophylaxis of an infection or colonizationby one or more microorganisms.
 16. The composition of claim 14 forsensitization or reduction in tolerance, of one or more microorganisms,to one or more anti-microbial agents.
 17. The composition of claim 15,wherein the one or more microorganisms are selected from the groupconsisting of Streptococcus pneumoniae, Campylobacter, Neisseriagonorrhoeae, Salmonella (including drug-resistant non-typhoidalSalmonella and drug-resistant Salmonella serotype typhi),Methicillin-resistant Staphylococcus aureus (MRSA), Shigella,Vancomycin-resistant Enterococcus (VRE), Vancomycin-resistantStaphylococcus aureus (VRSA), Erythromycin-resistant Group AStreptococcus, Clindamycin-resistant Group B Streptococcus,Carbapenem-resistant Enterobacteriaceae (CRE), drug-resistanttuberculosis, Extended spectrum Enterobacteriaceae (ESBL),multidrug-resistant Acinetobacter (including MRAB), Clostridiumdifficile, Enteropathogenic E. coli (EPEC), Pseudomonas aeruginosa, H.pylori, Streptococcus anginosus, and Uropathogenic E. coli (UPEC).
 18. Amedical device comprising the composition according to claim
 14. 19. Acoating comprising a composition according to claim 14, optionallywherein the composition is conjugated with one or more other agents. 20.An article comprising one or more surfaces having a coating according toclaim 19.